Vectors for molecule delivery to CD11b expressing cells

ABSTRACT

The invention relates to a novel use of a  Bordetella  adenylcyclase toxin in the manufacturing of vectors for targeting in vivo a molecule of interest, specifically to CD11b expressing cells. The invention also relates to an immunogenic composition that primes immune responses, to pharmaceutical compositions, and to a new vector for molecule delivery to CD11b expressing cells.

BACKGROUND OF THE INVENTION

The invention relates to a novel use of a Bordetella adenylcyclase toxinin the manufacturing of vectors for targeting in viva a molecule ofinterest, specifically to CD11b expressing cells. The invention alsorelates to an immunogenic composition that primes immune responses, topharmaceutical compositions and a new vector for molecule delivery toCD11b expressing cells.

Bordetella pertussis, the causative agent of whooping cough, secretesseveral toxins including the well-known pertussis toxin (PT) and theadenylate cyclase toxin (CyaA) or also adenylcyclase. CyaA is a criticalvirulence factor of B. pertussis in the murine respiratory model that isrequired for the early steps of lung colonization. Indeed, geneticdeletion of this toxin dramatically decreases the pathological effectsof B. pertussis infection, reducing the number of bacteria recoveredfrom the lung and almost abolishing the inflammatory cell recruitnentand the lung lesions observed after infection [Weiss et al., 1984; Weisset al., 1989; Gross et al., 1992; Khelef et al., 1992; Khelef et al.,1994; Gueirard et al., 1998]. Moreover, CyaA is an antigen protectiveagainst B. pertussis infection in the murine respiratory model [Guiso etal., 1989; Guiso et al., 1991].

Originally discovered by Hewlett et al in B. pertussis culturesupernatents [Hewlett et al., 1976], the adenylcyclase was later foundto be activated by the eukaryotic calmodulin [Wolff et al., 1980]. Thisstriking feature quickly found a rationale when it was shown by Conferand Eaton that the adenylcydase could enter into eukaryotic cells where,upon activation by calmodulin, it could trigger a large increase in cAMPwithin these target cells [Confer et al., 1982].

Adenylcyclase is encoded by the cyaA gene, and its expression, like thatof other virulence genes of B. pertussis, is coordinately regulated byenvironnemental signals. The cyaA gene is part of an operon that alsocontains genes cya B, D and E, that are required for CyaA secretion[Ladant et al., 1999].

The CyaA toxin is a bifunctional protein of 1706 residues that is madeof a N-terminal catalytic domain of 400 amino acids and a C-terminalpart of 1306 residues which is responsible for the binding of the toxinto target cell membrane and the subsequent delivery of the catalyticmoiety into the cell cytosol [Sakamoto et al., 1992] [Ladant et al.,1999]. This part also exhibits a weak hemolytic activity due to itsability to form cation-selective channels in biological membranes [Benzet al., 1994] [Gray et al., 1998]. This region is homologous toEscherichia coli hemolysin and other members of the RTX (Repeat inToXin) family of bacterial toxins. In particular, it contains a seriesof glycine and aspartate-rich nonapeptide repeats that are involved incalcium binding [Rose et al., 1995] [Coote et al., 1992].

The CyaA polypeptide is synthesized as an inactive protoxin that isconverted to an active toxin by posttranslational palmitoylation of twointernal lysines (lysines 856 and 963). This modification requires theproduct of an accessory gene, cyaC, which is located nearby cyaA on B.pertussis chromosome.

CyaA has been shown to bind to and invade a variety of cell typesincluding cells lacking membrane traffic like mammalian erythrocytes[Rogel et al., 1992]. This suggested that the catalytic domain of CyaAis directly translocated across the plasma membrane of target cells. Theinternalization of the catalytic domain into the cell cytosol is calciumand temperature-dependent and depends upon the plasma membrane potential[Rogel et al., 1992] [Karimova et al., 1998] [Otero et al., 1995].However, the molecular mechanisms by which the toxin transports itsN-terminus catalytic domain across the membrane remain largely unknownto date. Furthermore no specific receptor has been reported for CyaAbinding.

The physiological consequences of cellular intoxication by CyaA werecharacterized in vitro in phagocytes. Confer and Eaton first showed thatthe Cya A extracted from B. pertussis increases the intracellular cAMPlevel in neutrophils or macrophages leading to an inhibition ofchemotaxis and bactericidal functions such as superoxide generation andphagocytic abilities [Confer et al., 1982]. These activities were laterconfirmed with purified toxins or with bacterial mutants geneticallydeleted of CyaA [Pearson et al., 1987; Friedman et al., 1987] [Njamkepoet al., 2000]. On the contrary, and despite significant changes in theircAMP content, the viability of cell lines from non-hematopoietic originappeared to be unaffected by the CyaA intoxication [Bassinet et al.,2000]. Moreover, the present inventors previously demonstrated that B.pertussis CyaA can trigger macrophage apoptosis in vitro [Khelef et al.,1993; Khelef et al., 1995] and in vivo [Gueirard et al., 1998]. In thesemodels, genetic deletion of CyaA abolished macrophage apoptosis, but notneutrophil death, suggesting that CyaA i) is mainly responsible formacrophage apoptosis, ii) might be responsible for neutrophil apoptosis,but that another factor may also be responsible.

Besides that, in vivo studies performed in a murine model of B.bronchiseptica infection (the animal homologue of B. pertussis whoseCyaA is closely related) demonstrated that the major target of B.bronchiseptica CyaA toxicity is a GM-CSF-dependent andcyclophosphamide-sensitive population that controls the early steps ofinfection [Harvill et al., 1999]. These criterions identifiedneutrophils and possibly other cells induding macrophages or dendriticcells but no data is disclosed or suggested that the CD11b cell receptoris involved in the targeting by Cya A. These populations of target cellsfor CyaA is the same that limits the early phases of infection andfavors the development of an adaptative immune response that controlsthe latter phases of infection [Harvill et al., 1999].

Unlike other toxins, CyaA has been considered for a long time, asindependent of any receptor binding. This is based on the observationsthat i) CyaA can intoxicate a wide variety of model cell lines fromvarious origin [Ladant et al., 1999] ii) CyaA binds to Jurkat cells andsheep erythrocytes in a non saturable fashion [Gray et al., 1999].However, some specificity has been found in respect of cells infected byCyaA. Indeed, in Vivo studies showed that during murine respiratoryinfection with Bordetella species, CyaA destroyed specificallyleukocytes (especially macrophages) without damaging dramaticallyepithelial cells [Gueirard et al., 1998; Harvill et al., 1999].

It has been proposed in patent application WO 93/21324 to use therecombinant Bordetella adenylcyclase to induce a CD4+ T cell or a CD8+ Tcell response; however, since no specific receptor for Bordetellaadenylcyclase was identified, it was not known if the antigenpresentation was related to uptake by non professional antigenpresenting cell followed by cross-priming and presentation by dendriticcells or if the antigen was targeted to professional Antigen PresentingCells (pAPC).

In line with their surface phenotype, dendritic cells (high expressionof MHCI and II, costimulatory and adhesion molecules) represent the mostpotent APC in many in vitro assay for the priming of naive T cells [Bellet al., 1999; Viola et al., 1999]. Other APC like resting naive B cells,for example, could even be tolerogenic since injection of resting, maleB cells into female hosts leads to the specific tolerization ofmale-specific CD8+ T cells [Fuchs et al., 1992]. In vitro, naive B cellscould delete naive CD8+ T cells via a Fas dependent-mechanism [Bennettet al., 1998].

Moreover, Ag presentation by dendritic cells correlates in vivo with theinduction of T cell responses. This has been established forMHCII-restricted Ag presentation. Adjuvant-free Ag injection via theintravenous (iv) route usually does not induce T cell priming [Kyburz etal., 1993; Aichele et al., 1994; Aichele et al., 1995] and leads to Agpresentation by non-specific B cells [Guery et al., 1997) [Zhong et al.,1997; Reis e Sousa et al., 1999] and, eventually dendritic cells[Crowley et al., 1990] Zhong et al., 1997; Reis e Sousa et al., 1999].In contrast, local immunization strategies like subcutaneous (sc)immunization in the presence of adjuvant usually, induces T cell primingand targeted Ag presentation by Langerhans cell migrating from the skinto the LN draining the immunization site. In this case, B cells andmacrophages are not involved [Guery et al., 1996]. Similar results wereobtained after sc or intradermic (id) DNA immunization for MHCII andMHCI -peptide complexes: dendritic cells can be directly transfected atthe local site of injection and then migrate to the afferent LN viaafferent lymphatics [Condon et al., 1996; Casares et al., 1997; Porgadoret al., 1998]. The migration is known as a key event of immunity sincemechanical disruption of afferent lymphatics abrogates T cell responseto skin sensitizers or skin grafts [Zinkemagel et al., 1997].

Therefore, targeting to dentritic cells is essential for CD4⁺ and CD8⁺ Tcell stimulation. Since most antibody responses are dependent upon CD4⁺T cell help, targeting antigen to dendritic cells is a major goal invaccination.

Applicants have been interested in studying the presentation ofadenylcyclase of Bordetella species by T cell, and have identified aspecific receptor molecule present on specific cells, that interactswith CyaA and opens new possibilities for the use of CyaA as aproteinaceous vector for molecules of interest.

Genetically detoxified bacterial toxins represent candidates as vaccinevectors, in particular for T epitope, due to their ability to invadeeukaryotic cells (Ladant et al., 1999). However, few proteinaceousvectors were shown to prime CTL responses in vivo (Ballard et al., 1996,Cabonette et al., 1999). Moreover, despite numerous in vitro promisingstudies, no vector was shown to be exclusively targeted to pAPCparticularly to dendritic cells and more particularly to myeloiddendritc cells.

The inventors have further shown that other cells, especiallyneutrophils, could be targeted by the vectors of the present invention.

The invention provides means that may at least in part, fulfil theseneeds and proposes new vectors that would specifically target moleculesto determined populations of pAPC for example, to enable stimulatingimmune response.

Moreover, molecule targeted to the pAPC and specific leukocytes wouldenable the manufacturing of new vectors useful to deliver biologicallyactive molecule to the proximal environment of these cells. Thesemolecules, for example, could modulate the functional properties of thetargeted cells or those involved in the immune response or in theinflammatory response.

Indeed, the inventors have found that Bordetella pertussis adenylcyclasetoxin binds specifically with a cellular receptor designated(CD11b/CD18) α_(M)/β₂ receptor and that this interaction is required forthe intracellular delivery of the adenylcyclase domain to the cytosol ofcells and subsequent for cell death. α_(M)/β₂ (CD11b/CD18) integrin is adimer of the 2 integrin family, the expression of these integrins beingrestricted to leukocytes. CD11b/CD18 α_(M)/β₂ displays a pattern ofexpression in mouse and human, which is restricted toneutrophils/granulocytes, macrophages, dendritic cells, NK cells andsubsets of B and T CD8+ lymphocytes (Jeyaseelan et al., 2000, Amaout etal., 1990).

Therefore, this receptor would represent an ideal target for newvectors, designed in particular for T epitope immunization.

Applicants have shown in the present invention that the Bordetellaadenylcyclase can be used to target a molecule in vivo specifically toCD11b expressing cells.

In particular, Applicants have shown in the present invention that apeptide antigen comprised in the Bordetella pertussis adenylcyclasetoxin can efficiently be targeted specifically to the surface ofdendritic cells, translocated in the cytosol of said dendritic cells andprime a CTL response.

In a specific embodiment, said response is obtained bypassing adjuvantrequirement and CD4+ T-cell help.

It has also been shown that genetically modified adenylcylase can bechemically coupled to a peptide of interest to target said peptide toCD11b expressing cells, especially the cytosol of dendritic cells.

This invention thus provides new efficient immunogenic composition aswell as new drug delivery vector to CD11b expressing cells.

DETAILED DESCRIPTION OF THE INVENTION

This invention relates to the use of Bordetella adenylcyclase in themanufacturing of a proteinaceous vector for targeting a molecule ofinterest specifically to CD11b expressing cells.

The invention also relates to the use of a Bordetella adenylcyclasewherein said adenylcyclase is recombined with an antigen and especiallymodified by insertion of a peptide of interest or modified by insertionof a molecule of interest for the preparation of a composition for thetargeting of said peptide or molecule specifically to CD11b expressingcells.

The term “specifically” means within the context of the presentinvention that the adenylcyclase when used as a vector for a molecule ofinterest, is preferentially directed to CD11b expressing cells, therebyoffering means to target the molecule of interest at the surface of saidcells or within said cells in a selective way with respect to othercells.

In one embodiment of the invention, the molecule of interest isessentially directed to CD11b expressing cells.

As used herein, the term “CD11b expressing cells” relates to the cellsthat express the CD11b/CD18 α_(m)/β₂ receptor on their surface. Inparticular, these cells are granulocytes/neutrophils, macrophages, NKcells, subsets of T CD8+ and B cells and myeloid dendritic cells.

Since CD11b expressing cells and more specifically the myeloid dendritccells, the neutrophils and the macrophages are involved in essentialfunctions of the immune and innate defence system, in particular ininflammatory and specific immune responses, the invention relates to themanufacturing of a proteinaceous vector or a composition capable oftargeting a molecule or a peptide of interest to these CD11b expressingcells especially to myeloid dendritic cells, neutrophils or macrophages.

In particular, in one embodiment, the targeting of said molecule orpeptide is effective in vivo.

The invention thereby provides means appropriate for the design ofcompositions suitable for administration to animal or human hostsrequiring targeting of certain leukocytes and in particular myeloiddendritic cells, neutrophils or macrophages.

Bordetella adenylcyclase is the calmodulin-dependent adenylcyclasesecreted in Bordetella species or fragment thereof, said fragmentretaining the function properties of the adenylcyclase, a majorvirulence factor mandatory for the initial phases of bacterialcolonization in the lung. The adenylcyclase is synthesized and secretedin the form of a polypeptide of 1706 amino acids: Thecalmodulin-dependent catalytic activity is localized in the first 400amino acids. In order to be active, said adenylcyclase toxin is renderedinvasive and hemolytic when post-traductionally modified by thecoexpression of the cyaC gene product.

The following specific features of Bordetella adenylcyclase toxinindicate that this toxin can be used in the manufacturing of aproteinaceous vector for targeting in vivo a molecule of interest toCD11b expressing cells:

-   -   a) this adenylcyclase binds specifically to CD11b expressing        cells    -   b) the N-terminal catalytic domain is translocated into the        cytosol of those CD11b expressing cells.    -   c) the C-terminal domain binds to the membrane of CD11b        expressing cells and could internalize by endocytic pathway.    -   d) epitope chemically coupled to genetically modified        adenylcyclase can elicit in vivo specific CTL responses.

The expression “adenylcyclase” encompasses, within the presentinvention, natural or modified adenylcyclase, including genetically orchemically modified adenylcyclase, providing the resulting product isable to target a molecule of interest specifically to CD11b expressingcells.

The invention thus relates to the use of Bordetella adenylcyclase asdefined above and more particularly to the use of a modified orrecombinant, adenylcyclase for targeting a molecule of interestspecifically to CD11b expressing cells.

More specifically, recombinant adenylclases include adenylcyclases whichhave been genetically modified to provide either adenyclases withpeptide sequence or cysteine residues inserted within the catalyticdomain, or truncated adenylcyclases lacking all or part of theircatalytic domain.

Due to the specific interaction between Bordetella adenylcyclase toxinand CD11b /CD18 α_(m)β₂ receptor, the molecule of interest isspecifically targeted at least to the surface of CD11b expressing cells.In a particular embodiment of the invention, the Bordetellaadenylcyclase toxin is used in the manufacturing of a proteinaceousvector to deliver the molecule of interest either in the cytosol ofCD11b expressing cells, at the surface of CD11b expressing cells or intothe endocytic pathway of CD11b expressing cells.

Expression vectors for the preparation of recombinant Bordetellaadenylcyclase are described in patent application WO 93/21324 (InstitutPasteur). Novel expression vectors for the preparation of geneticallymodified Bordetella adenylcyclase appropriate for chemical coupling of apeptide of interest are also described in the experimental parthereafter. More specifically, expression vectors may be constructeddirecting the expression of both the cyaA gene and the cyaC gene (Seboet al., 1991). In parallel, a secondary plasmid carrying genes necessaryfor the secretion of the cytotoxic adenylcyclase in E. coli, such ashlyB and hlyD as described for example in Meckman et al., 1985 can beconstructed. In particular, the expression plasmid pCACT3 described inWO 93/21324 can be used. Using this plasmid, the adenyclyclase may beexpressed in E. coli and possibly secreted by this bacterium in largeamounts. It is also readily purified for example using affinitychromatography on CaM Affi-Gel resin or other published procedures, asthose using DEAE-sepharose and phenylsepharose (Guermonprez et al.,2000).

In one embodiment of the invention, the adenylcyclase is a recombinantgenetically modified adenylcyclase. In particular, mutations such aspoint mutations, deletions or insertions can be obtained using usualsite-directed or random mutagenesis techniques, provided that thedomains necessary for binding to CD11b expressing cells and, optionnalyfor translocation in the cytosol are still functional. Assays toevaluate specific binding of recombinant toxins and fragments thereof,to CD11b expressing cells and optionnaly subsequent translocation of thecatalytic domain are described in the following experimental part.

In another embodiment of the invention, the recombinant Bordetellaspecies adenylcyclase is a fragment of the native, modified orrecombinant Bordetella species adenylcyclase toxin, wherein saidfragment is capable of binding the CD11b receptor. In particular, it hasbeen found in the present invention that fragment encompassing residues373 to 1706 (CyaA 373-1706) contains the structures essentially requiredfor the interaction with the CD11b/CD18 receptor. Thus, a preferredfragment of the Bordetella species adenylcyclase toxin is the Bordetellaadenylcyclase toxin lacking all or part of the N-terminal catalyticdomain, and more specifically Bordetella pertussis adenylcyclase lackingall or part of residues 1-373.

Specific binding to CD11b can be assessed in vitro with anti-CD11bmonoclonal antibodies as illustrated in the examples.

In order to be used in the manufacturing of proteinaceous vector or thepreparation of a composition, the adenylcyclase is preferably non toxic.Non toxic mutants of the adenylcyclase toxin are well described in theArt. (Betsou et al., 1993; Betsou et al., 1995.

In a preferred embodiment of this invention, the adenylcyclase isisolated from Bordetella pertussis.

In specific embodiments, the molecule of interest is selected in thegroup comprising: peptides, glycopeptides, lipopeptides,polysaccharides, oligosaccharides, nucleic acids, lipids and chemicals.

In specific embodiments, a molecule of interest is a heterologousantigen. As used herein, the term “heterologous” refers to an antigenother than the adenylcyclase which is used in the vector itself.

In a preferred embodiment of the invention, the manufacturing of theproteinaceous vector comprises the step of inserting a heterologousmolecule and especially a peptide in the catalytic domain of theadenylcyclase at a permissive site.

As used herein, the term “permissive site” relates to a site where theheterologous molecule and especially a peptide can be inserted withoutsubstantially affecting the desired functional properties of theadenylcyclase toxin, i.e. without affecting the domains necessary forthe specific binding to CD11b /CD18 receptor and advantageously withoutaffecting the process of translocation of the catalytic domain. In apreferred embodiment, the capacity of the CyaA toxin to promote thesynthesis of cAMP in the targeted cells is further maintained.

Methods to select for permissive sites are presented for example inWO93/21324 and in Ladant et al., 1992. In particular, a methodologyusing a double selection (resistance to an antibiotic and calorimetrictest on dishes by α-complementation) enables to identify readilyoligonucleotides insertions (which preserve the reading frame) in theportion of the gene coding for the N-terminal catalytic domain of thetoxin. The functional consequences of these mutations on the catalyticactivity of the toxin may be readily analysed, both genetically(functional complementation of an E. coli cya³¹ strain) andbiochemically (characterization of the stability of the modifiedadenylcyclases, of their enzymatic activity, of their interaction withcaM, etc.). This methodology has enabled a large number of mutations tobe screened in order to identify the sites which are potentiallyadvantageous for the insertion of antigenic determinants.

In specific embodiments of the invention, a permissive site is selectedfrom the group consisting of residues 137-138, residues 224-225,residues 228-229, residues 235-236 residues 317-318 and residues 335-336of the Bordetella pertussis adenylcyclase.

However, other permissive sites may be used in the present invention,that can be identified for example by use of the methodology indicatedabove, especially sites between residues 400 and 1700.

The manufacturing of the proteinaceous vector can also comprise a stepof fusing a molecule of interest, for example a heterologous peptide, atthe N-terminal extremity of a Bordetella adenylcyclase lacking all orpart of its N-terminal catalytic domain, and more preferably, Bordetellapertussis adenylcyclase lacking residues 1-373.

In a preferred embodiment of the invention, the adenylcyclase accordingto one of the above definitions is used in the manufacturing of aproteinaceous vector or in the preparation of a composition specificallydesigned to prime CD8+ cytoxic T-cell response (CTL response) saidresponse follows the targeting of the adenylcyclase modified (especiallyrecombined or conjugated) with a molecule of interest to CD11bexpressing cells, followed by the translocation of the molecule ofinterest to the cytosol of said CD11b expressing cells, and inparticular to myeloid dendritic cells. In this context, the molecule ofinterest is or comprises preferably an epitope or an antigen.

As used herein, the term “epitope” refers to a heterologous molecule andespecially a heterologous peptide that can induce an immune response.

In specific embodiments, the antigen is selected from the groupconsisting of an intracellular bacterial cell antigen, a tumoral cellantigen, a viral antigen, a fungus antigen or a parasite cell antigen.

In a preferred embodiment of the invention, the adenylcyclase, i.e., anatural, modified or recombinant adenylcyclase according to one of theabove definitions is used in the manufacturing of the proteinaceousvector or in the preparation of a composition specifically designed toprime CD4+ cells response said response follows the targeting of theadenylcyclase modified (especially recombined or conjugated) with amolecule of interest to CD11b expressing cells, in particular myeloiddendritic cells. In this context, the molecule of interest is orcomprises preferably an epitope or an antigen.

A molecule of interest can be especially an antigen selected from thegroup consisting of: a poliovirus antigen, an HIV virus antigen, aninfluenza virus antigen, a choriomeningitis virus epitope, a tumorantigen.

The functional properties of the CD11b expressing cells definefurthermore a novel use of the Bordetella adenylcyclase toxin in themanufacturing of a proteinaceous vector for drug targeting to thesespecific cells. In this context, in one specific embodiment of theinvention, the so-called molecule of interest is a drug Said drug may bechemically or genetically coupled to the adenylcyclase. Method forcoupling a drug to a polypeptide are well known in the Art and comprisefor example disulfide linkage by using N-pyridyl sulfonyl-activatedsulfhydryl.

Advantageously, a molecule of interest is an anti-inflammatory drugwhich is, when coupled to the adenylcyclase toxin, specifically targetedto the surface of the cells involved of the inflammatory response, suchas neutrophils.

In particular, it is shown for the first time in the Experimental partthat it is possible to graft molecules to CyaA by a chemical linkage orby genetic insertion for in vivo targeting to CD11b+ antigen presentingcells and particularly to the cytosol of CD11B+ antigen presentingcells. Indeed, when coupling a molecule corresponding to a given CD8+T-cell epitope to the catalytic domain of detoxified CyaA, either bymeans of a disulfide bond or by genetic insertion, it has been foundthat the engineered molecule can elicit in vivo specific CTL response,thereby showing that said CD8+ T-cell epitope is translocated into thecytosol of CD11b -expressing cells.

More specifically, antigen presentation for selective CD8+ cytotoxiccells priming is mainly performed by myeloid dendritic cells.

Accordingly, in a specific embodiment, the recombinant adenylcyclaseused for the manufacturing of proteinaceous vector is a geneticallymodified adenylcyclase containing one or more molecule(s) chemicallycoupled by means of a disulfide bond to genetically inserted cysteineresidue(s) located within the catalytic domain of said adenylcyclase.

Indeed, multiple molecules can be chemically coupled to theadenylcyclase by means of a disulfide bond to different cysteineresidues located at different permissive sites within the catalyticdomain.

Applicant has also shown that CTL specific for the vectorized antigencan be primed in vivo after a single intravenous injection of therecombinant toxin, especially with no need to provide an heterologousadjuvant. These results shown in the experimental part and in particularthe specific targeting of the epitope to myeloid dendritic cells enablenew immunization strategies that bypass the requirement for adjuvant andCD4+ T cell help.

Therefore, the invention also relates to the use of a Bordetellaadenylcyclase toxin recombined with a molecule and especially a peptideof interest for the preparation of a composition formulated forintravenous administration and enabling a CD8+ T cell immune response invivo, said composition being free of a heterologous adjuvant. Theinvention also concerns this composition as such.

The invention in particular also relates to a new immunogeniccomposition formulated for administration, especially intravenousadministration, in an animal or human host, characterized in that itcomprises a recombinant Bordetella adenylcyclase which comprises anantigen inserted in the catalytic domain.

The invention further relates to a pharmaceutical composition foradministration in a human or an animal formulated for targeting amolecule of interest specifically to CD11b expressing cellscharacterized in that said molecule of interest is coupled to aBordetella species adenylcyclase.

In one preferred embodiment, the molecule of interest is selected in thegroup comprising: peptides, glycopeptides, lipopeptides,polysaccharides, oligosaccharides, nucleic acids, lipids and chemicals.

In another preferred embodiment, the molecule of interest is an antigen.

In another specific embodiment, the pharmaceutical or immunogeniccomposition comprises a nucleic acid construction encoding therecombinant Bordetella species adenylcyclase comprising a recombinantBordetella species adenylcyclase coupled to a molecule of interest

In specific embodiments, the adenylcyclase is from Bordetella pertussistoxin.

In other specific embodiments, the adenylcyclase toxin is a geneticallymodified toxin. In one preferred embodiment, the adenylcyclase is a nontoxic adenylcyclase, especially a detoxified adenylcyclase.

In one preferred embodiment, the genetically modified adenylcyclase isable to translocate the molecule of interest specifically in the cytosolof CD11b expressing cells.

In particular, said genetically modified adenylcyclase is a Bordetellaadenylcyclase lacking all or part of its catalytic N-terminal domain,and more specifically Bordetella pertussis adenylcyclase lackingresidues 1-373.

Advantageously, the genetically modified adenylcyclase comprises one ormore cysteine residues inserted within the catalytic domain atpermissive sites. Such genetically modified adenylcyclase can be coupledto one or more molecule(s) of interest by means of disulfide bond(s) atthe inserted cysteine residue(s).

In a preferred embodiment, the molecule, and especially an antigen isinserted in the permissive sites selected from the group consisting ofresidues 137-138, residues 224-225, residues 228-229, residues 235-236and residues 317-318, and residues 335-336 of the Bordetella pertussisadenylcyclase, or the molecule is fused to the N-terminal part of aBordetella adenylcyclase lacking all or part of its N-terminal catalyticdomain, and more specifically to the N-terminal part of a Bordetellapertussis adenylcyclase lacking residues 1-373.

In a specific embodiment, the molecule is an antigen which is anintracellular bacterial cell antigen, a tumoral cell antigen, a viralantigen, a fungus antigen or a parasite cell antigen.

In preferred embodiments, a molecule of interest is an antigen selectedfrom the group consisting of: a poliovirus epitope, an HIV virus, aninfluenza virus antigen, a choriomeningitis virus antigen, a tumorantigen.

In another embodiment, the genetically modified toxin is able to deliverthe molecule of interest specifically at the surface of CD11b expressingcells or into the endocytic pathway.

The applicants have shown that in vivo intravenous administration of theimmunogenic composition in an animal or human host as defined in thepresent invention without adjuvant(s) is sufficient to promoteefficiently an immune response in said animal or human host.

In particular, the immunogenic compositions of the invention are capableof inducing or stimulating, in vivo or in vitro an immune cell responseinvolving specifically dendritic cells.

As a consequence, in a specific embodiment, the immunogenic orpharmaceutical composition is advantageously devoid of priming adjuvantscommonly used in the Art, such as aluminium hydroxide.

In one specific embodiment, the molecule of interest is a drug,preferably an anti-inflammatory drug.

Furthermore, the invention also relates to the use of the immunogeniccomposition as defined above for the preparation of a vaccine or animmunotherapeutic composition, for administration to an animal or humanhost.

As used herein, the term “immunotherapeutic composition” relates to acomposition which leads to an immunological response and which isassociated to therapeutic treatments, such as treatment against cancers,viral infections, parasites infections or bacterial infections.

The invention further relates to a method to immunize an animal or humanhost, wherein said method comprises the steps of:

-   -   a) providing an immunogenic composition as defined above;    -   b) administering said immunogenic composition, preferably via        intravenous route, to said host in order to promote an immune        response.

The invention finally relates to a proteinaceous vector for delivery ofa molecule of interest, specifically to CD11b expressing cells,characterized in that said vector comprises a Bordetella speciesadenylclyclase and more preferably a recombinant or modified Bordetellaspecies adenylcyclase coupled to said molecule of interest.

The proteinaceous vector is able to target a molecule of interest toCD11b expressing cells via the specific binding of Bordetellaadenylcyclase with CD11b/CD18 α_(m)β₂ which is present on the surface ofspecific cells. In one specific embodiment, the vector is also able todeliver the molecule of interest specifically in the cytosol of CD11bexpressing cells.

In specific embodiments, the proteinaceous vector is able to target amolecule of interest to dendritic cells, particularly myeloid dendriticcells, or to neutrophils. The molecule of interest is chemically orgenetically coupled to the adenylclase, more preferably the recombinantadenylcyclase toxin. Method for coupling a molecule to a polypeptide arewell known in the Art. As an example, the inventors showed that a biotinderivative, biotin HDPD can be selectively coupled on an unique cysteineresidue genetically inserted within the catalytic domain. Syntheticpeptides have been coupled similarly to the cysteine containingadenylcyclase toxin, or to genetically modified adenylcyclase devoid ofnative cysteine residue but containing an unique genetically insertedcysteine residue.

In another specific embodiment, a molecule of interest coupled to theadenylcyclase is an epitope chemically coupled to a genetically modifiedadenylcyclase devoid of native cysteine residue but containinggenetically inserted cysteine residue(s) at a permissive site within itscatalytic domain.

In a specific embodiment, the proteinaceous vector comprises agenetically modified adenylcyclase. In one preferred embodiment, theadenylcyclase is a recombinant non toxic adenylcyclase from Bordetellaperitussis.

The invention more specifically relates to a proteinaceous vector whichconsists of a recombinant Bordetella adenylcyclase lacking all or partof the N-terminal catalytic domain, and more preferably a Bordetellapertussis lacking residues 1-373.

Another preferred proteinaceous vector of the invention is a geneticallymodified Bordetella adenylcyclase devoid of native cysteine residue, butcontaining genetically inserted cysteine residue(s) at a permissive sitewithin the catalytic domain.

Advantageously, in one specific embodiment of the invention, a drug tobe delivered is an anti-inflammatory drug which is, when coupled to theadenylcyclase toxin, targeted to the surface or to the cytosol of thecells involved of the inflammatory response, such as neutrophils.

The following examples will illustrate the present invention withoutlimiting the scope of the claimed invention. More specifically, in partA, experimental data are shown that reveal the specific binding ofadenylcyclase toxin to CD11b/CD 18 receptor, and in particular thespecific binding of adenycyclase toxin to CD11b expressing cells invitro. In part B, the experimental results show the possibility totarget a genetically coupled molecule, more particularly an antigen invitro and in vivo specifically to the cytosol of CD11b expressing cells,and in particular to myeloid dendritic cells. Moreover, the results showthat the targeting is mediated by the CD11b receptor and that CTLpriming is observed after systemic immunization in the absence ofadjuvant. In part C., it is shown that specific coupling of epitopepeptides by means of a disulfide bond on CyaA fragments can be carriedout to provide new proteinaceous vectors. In part D., it is shown that,only the C-terminal part of CyaA is necessary and sufficient forinteraction with CD11b receptors.

Finally, the results indicate that, unlike many other CTL responses asthose raised against cross-priming antigen, CD4+ T cell help was notmandatory for priming of CTL responses.

LEGENDS TO FIGURES

FIG. 1: Saturable binding of CyaA correlates with CD11b expression

a: CyaA binding at the surface of macrophages (J774A.1), B cells(LB27.4), and T cells (EL4) was performed at 37° C. for 20 minutes.Surface-bound CyaA was detected with a biotinylated anti-CyaA polyclonalantibody, revealed by streptavidin-PE and detected by flow cytometry onliving cells, as described in the material and methods section. Bindingis expressed as ΔMFI=(mean fluorescence intensity value of cellsincubated with CyaA)−(mean fluorescence intensity of cells withoutCyaA).

b, c, d, e: Surface expression of β₂ integrins on J774A.1, LB27.4 andEL4 cells. CD11a (b), CD11b (c), CD11c (d), and CD18(e) expression weredetermined by flow cytometry using specific mAbs coupled to PE. Integrinexpression is expressed as ΔMFI=(mean fluorescence intensity value ofcells stained with specific mAb)−(mean fluorescence intensity of cellsstained with an isotype control mAb).

FIG. 2: CyaA binding to murine cell lines is blocked by anti-CD11b mAb.

Cells were preincubated at 4° C. for 15 minutes with or without 20 μg/mlof specific mAbs and then incubated at 4° C. for 20 minutes with 5 μg/mlCyaA and with 10 μg/ml of specific mAbs if present during thepreincubation. Surface-bound CyaA was detected with a biotinylatedanti-CyaA polyclonal, revealed by streptavidin-PE and detected by flowcytometry on living cells, as described in the material and methodssection.

a, b: Effect of the M1/70 anti-CD11b mAb on the binding of various dosesof CyaA. FSDC dendritic cells (a) or J774A.1 macrophages (b) werepreincubated with medium alone (∘) or with M1/70 anti-CD11b mAb (●) andthen incubated with CyaA with or without M1/70 anti-CD11b mAb. Bmax wasdetermined by fitting experimental points obtained from experimentsperformed without mAbs to ΔMFI=Bmax*[CyaA]/(K_(d)+[CyaA]). Binding ofCyaA is plot as a % of Bmax plot against CyaA concentration.

c, d: Effect of specific mAbs on a fixed dose of CyaA binding. FSDC (c)or J774A.1 (d) cells were preincubated with or without specific mAbs(anti-CD11a, 2D7, antiCD11b, M1/70 and 5C6, anti-CD11c, HL3, anti-CD18,C17/16, control A95-1) and incubated with CyaA at the fixedconcentration of 5μg/ml. Values of ΔMFI obtained for CyaA binding oncells treated with specific mAbs were normalized as ΔMFI values obtainedfor CyaA binding without mAb.

FIG. 3: CyaA binding to the human neutrophils is blocked by anti-CD11band anti-CD18 mAbs.

a, b, c: Fluorescence histograms of freshly purified neutrophils werepreincubated with medium alone (a), the 44 anti-CD1 b mAb (b) or anisotype-matched control mouse mAb (c) and then incubated with (gray) orwithout (blank) biotinylated CyaA and revealed by streptavidine-PE. Cellnumber is ploted against log of PE fluorescence.

d: Effect of specific mAbs on CyaA binding to neutrophils (anti-CD11b,44, M1/70, anti-CD18, TS/18, control mouse IgG2a, control rat IgG2b,A95-1). Freshly purified neutrophils were preincubated with or withoutspecific mAbs and incubated with CyaA. Values of ΔMFI obtained for CyaAbinding on cells treated with specific mAbs were normalized as a % ofthe ΔMFI values obtained for CyaA binding without mAb.

FIG. 4: Intracellular cAMP increase and cell death mediated by CyaA arespecifically blocked by an anti-CD11b mAb in J774A.1 cells.

a: Effect of specific mAbs on intracellular cAMP accumulation. J774A.1cells were preincubated at 4° C. for 1 h with or without 10 μg/ml ofspecific mAbs (anti-CD11b, M1/70, anti-CD18, C17/16) and then incubatedat 37° C. for 20 min with 5 μg/ml CyaA and with 10 μg/ml mAbs if presentduring the preincubation. Intracellular cAMP contents were determined asdescribed in the materials and methods section.

b: Effect of specific mAbs on CyaA mediated cell death. J774A.1 cellswere preincubated at 4° C. for 1 h with medium alone or with 10 μg/ml ofspecific mAbs (anti-CD11a, 2D7 anti-CD11b, M1/70, anti-CD11c, HL3,anti-CD18, C71/16, control, 2.4G2). Then they were incubated at 37° C.for 2 h with 0.5 μg/ml CyaA and with 10 μ/ml of specific mAbs whenpresent during the preincubation. Cell lysis was determined by LDHrelease using the Cytotox 96™ assay.

FIG. 5: CHO cells bind CyaA and become sensitive to CyaA whentransfected with CD11b, but not with CD11c

a, b: CyaA binding at the surface of CHO transfectants. CHO cellstransfected with human CD11b/CD18 (●), human CD11c/CD18 (∘) ormock-transfected (

) were incubated with various doses of CyaA for 20 min at 37° C. (a) or4° C. (b). Surface-bound CyaA was detected with a biotinylated anti-CyaApolyclonal antibody, revealed by streptavidin-PE and detected by flowcytometry on living cells, as described in the material and methodssection. Binding is expressed as ΔMFI=(mean fluorescence intensity valueof cells incubated with CyaA)−(mean fluorescence intensity of cellswithout CyaA).

c: Intracellular cAMP accumulation in CHO transfectants. CHO cellstransfected with human CD11b/CD18 (●), human CD11c/CD18 (∘) ormock-transfected (

) were incubated with or without CyaA for 20 min at 37° C. IntracellularcAMP contents were determined as described in the materials and methodssection.

d: Cell lysis in CHO transfectants. CHO cells transfected with humanCD11b/CD18, human CD11c/CD18 or mock-transfected were incubated with 5μg/ml CyaA for 4 h at 37° C. Cell lysis was determined by LDH releaseusing the Cytotox96™ assay.

FIG. 6: Intravenous immunization with CyaAOVA primes anti-OVA CTLresponses in a B cell, CD4 and CD40-independent way.

C57BL/6 WT +/+ (a), CD4−/− (b), CD40−/− (c) or IgM−/− (d) mice wereintravenously immunized with 50 μg of CyaAOVA, a genetically detoxifiedform of CyaA carrying the H-2K^(b) restricted SIINFEKL epitope from OVA(●, ∘) or with CyaAE5, a control detoxified toxin without the OVAepitope (▴, Δ). Seven days after, animals were sacrified and splenocyteswere restimulated in vitro for 5 days with 10 μg/ml of the pOVAsynthetic peptide in the presence of irradiated C57BL/6 splenocytes. CTLactivity was assessed in a 4 hours chromium⁵¹ release assay againstH-2K^(b+) EL4 cells previously pulsed (●,▴) or not (∘, Δ) with pOVA at10 μg/ml.

FIG. 7: Identification of splenic antigen presenting cells involved inCyaAOVA presentation in vitro or in situ, after intravenousimmunization.

The low density fraction of splenocytes presents CyaAOVA to a specificanti-OVA CD8+ T cell hybridoma (a, b):

In vitro assay (a): Low (LDF, ●) and high density (HDF, ▴) fractions orunfractionated total splenocytes from naive mice (TSC, ▪, □) werecultured with B3Z, a CD8⁺ T cell hybridoma specific for the pOVA peptidein the context of H-2K_(b). After 18 hours of coculture in the presenceof the recombinant detoxified CyaA carying the OVA peptide (CyaAOVA, ●,▴, ▪) or a control peptide (CyaALCMV, □) at various concentrations, IL-2released in supernatants was measured in a CTLL proliferation assay.Results are expressed in Δcpm and ploted against CyaA concentrationduring the assay Δcpm=[cpm+CyaA]-[cpm−CyaA]).

Ex vivo assay (b): Spleen cells were obtained from mice previouslyimmunized iv (6-12 hours) with 50 μg of CyaAOVA (●, ▴, ▪) or CyaALCMV(∘) and fractionated in LDF and HDF. Various numbers of cells recoveredfrom TSC (▪), LDF (●, ∘) or HDF (▴) or unfractionated splenocytes (▪)were directly put in culture with B3Z without addition of recombinantCyaA. IL-2 release was assessed after 18 hours of culture as describedbefore. Results, expressed in cpm, are ploted against the number of APCpresent in each well.

Dendritic cells (CD11c⁺) are more efficient APC for CyaAOVA than theCD11b ^(high+) CD11c⁻ cells or B cells (CD45R⁺) (c,d):

In vitro assay (c): CD11c⁺ (●) sorted cells from LDF, CD11b ^(high+)CD11c⁻ (∘) and CD45R⁺ (▪) sorted cells from TSC, were put in culturewith B3Z for 18 hours in the presence of various concentrations ofCyaAOVA. IL-2 was assessed as above.

Ex vivo assay (d): Cells sorted by flow cytometry from C57BL/6 micepreviously (6-12 hours) immunized with 50 μg of CyaAOVA were used asAPC. CD11c+ (●), CD11b^(high+) CD11c⁻ (∘), CD45R⁺ (▪) sorted cells fromlow density splenocytes were directly put in culture for 18 hours withB3Z at various numbers of cells per well, without addition of CyaAOVA.IL-2 was assessed as above.

The CD8α⁻ myeloid dendritic cell subset is a more efficient APC for CyaAthan the CD8α⁺ lymphoid dendritic cell subset (e, f):

CD11c+ low density cells from naive mice (e) or mice previously (6-12hours) immunized iv with 50 μg CyaAOVA (f) were fractionated in myeloiddendritic cells (CD11c⁺ CD8α⁻,●) and lymphoid dendritic cells (CD11cCD8α⁺,∘) by flow cytometry and used as APC in in vitro (e) and ex vivo(f) assays for B3Z stimulation. IL-2 was assessed as above.

B cell genetic depletion does not impair CyaAOVA presentation bysplenocytes (g, h):

TSC (▪, □), LDF (●, ∘) or HDF (▴, Δ) from C57BL/6 WT mice (▪, ●, ▴) or Bcell deficient (□, ∘, Δ) were used as APC in an in vitro assay (g, ▪, □)or an ex vivo assay (h, ▪, ●, ▴, □, ∘, Δ) for B3Z stimulation as in a.Mice were either from naive (g) or previously (1.5 hours) immunized ivwith 50 μg CyaAOVA (h). IL-2 was assessed as above.

FIG. 8: The presentation of CyaAOVA by dendritic cells requires the TAPtransporters in vitro and in vivo after intravenous immunization.

In vitro assay (a): TSC (▴, Δ) or CD11c⁺ (●, ∘) sorted cells fromcontrol C57BL6 TAP+/+ (▴, ●) or TAP−/− mice (Δ, ∘) were cultured withB3Z in the presence or not of various doses of CyaAOVA. IL-2 wasassessed as described in FIG. 6 a. Results are expressed in cpm plotedagainst antigen concentration.

Ex vivo assay (b): TSC (▴, Δ) or CD11c⁺ (●, ∘) sorted cells from controlC57BL6 TAP+/+ (▴, ●) or TAP−/− mice (Δ, ∘) previously iv immunized with50 μg of CyaAOVA were cultured with B3Z for 18 hours. IL-2 was assessedas described in FIG. 6 a. Results are expressed in cpm ploted againstthe number of cocultured cells.

FIG. 9: Role of α_(M) β₂ integrin (CD11b) in CyaAOVA binding to cells.

Binding of CyaAOVA-biotine to TSC is blocked by anti-CD11b (a): TSCsuspensions were incubated at 4° C. with 10 μg/ml of the anti-CD11bM1/70 mAb or an isotype control mAb or nothing. Then, CyaAOVA-biotine at2 μg/ml (left pannels) or various concentrations (right pannel) wasadded to the cells for 30 mn at 4° C. After a wash, CyaAOVA-biotine wasrevealed with streptavidine-PE for 30 mn (Strep-PE). Then, afterwashing, cells were resuspended in PBS containing propidium iodide. Thesize (FSC) of living cells gated by propidium iodide exclusion wasplated against the Strep-PE fluorescence. The percentage of leukocytespositive for CyaAOVA-biotine was ploted against CyaAOVA-biotineconcentration during the staining.

Binding of CyaAOVA-biotine to low density cells correlates with theexpression of CD11b (b): LDF were triple stained for CD11c, CD8α andCyaOVAbiotne (or medium) or, in separate experiments with CD11c, CD8αand CD11b (or a control mAb). After a wash, cells were stained for 30 mnwith Strep-PE to reveal CyaAOVA-biotine, anti-CD11c-FITC andanti-CD8α-APC. Gates were done on iymphoid DC (CD11c⁺ CD8α⁺), myeloid DC(CD11c⁺ CD8α⁻), CD8+ T cells (CD11c⁻CD8α⁺) and other cells(CD11c⁻CD8α⁻). For each gate, CyaAOVA-biotine staining or CD11b stainingis ploted against cell number in separate histograms. Left histograms:LDF suspensions were incubated with 0 (histograms filled in grey), 2.5(narrow, open histograms) or 10 μg/ml (thick, open histograms) ofCyaAOVA-biotine for 30 minutes at room temperature. Right histograms:isotype control-PE (histogram filled in grey), CD11b-PE (thin, openhistograms).

FIG. 10: Role of α_(M) β₂ integrin (CD11b) in CyaAOVA presentation byMHCI

In vitro antigen presenting assay with TSC (a, b): The same experimentsthan in a, b were performed with TSC from naive C57BL/6 mice as APC. Thestimulation of B3Z was assessed by IL-2 release in coculturesupernatants measured in a CTLL proliferation assay. Results are plotedin cpm against CyaAOVA or pOVA concentration.

Ex vivo antigen presenting assay with TSC or CD11b⁺ and CD11b⁻ fractions(c, d): C57BL/6 mice were intravenously immunized with 50 μg of CyaAOVA(c) or 10 μg of pOVA (d). CD 11b⁺ (●) and CD11b⁻ (∘) cells were sortedby flow cytometry from TSC (Δ) and put in culture at various cell numberper well with B3Z. After 18 hours of coculture, the stimulation of B3Zwas assessed by IL-2 release. Results, expressed in cpm, are plotedagainst the numbers of APC from immunized animals present in each well.

FIG. 11: Summary of the methodology for chemical coupling of epitopes torecombinant CyaA through disulfide bond.

FIG. 12: A diagram of a vector for chemical coupling of CTL epitopes

FIG. 13: A graph showing IL-2 release by B3Z measured in a CTLLproliferation assay.

3.10⁵ spleen cells from C57BI/6 mice were cocultured for 18 h with 105B3Z cells in the presence of various concentrations of cyclases. The IL2release by B3Z was measured in a CTLL proliferation assay.

FIG. 14: A graph showing cytotoxic activity measured on ⁵¹Cr-labelledEL4 target cells pulsed (A) or not (B) with 50 μM of the OVA peptide.

C57BL/6 mice were iv injected with 50 μg of the various CyaA. Seven dayslater, spleen cells were in vitro stimulated with OVA peptide. Thecytotoxic activity was measured on 51 Cr-labelled target cells.

The FIG. 14 shows the in vivo capacity of the proteinaceous vectors ofthe invention to induce OVA-specific CTL responses.

FIG. 15: Inhibition of CyaA binding to CD11b by CyaA-E5 and CyaAfragments

CHO cells transfected with CD11b/CD18 were preincubated on ice for 1hour with different concentrations of CyaA-E5 (black triangle), CyaA1-383 (black square) or CyaA-373-1706 (Black diamond) and then incubatedon ice for 30 min with 5 μg/ml of biotinylated CyaA-E5. Surface boundcyclase was revealed using streptavidin-PE and analyzed by flowcytometry on living cells. Results are expressed as mean fluorescenceintensity (A), percentage of positive cells (B) and percentage ofinhibition (C).

FIG. 16: Schematic representation of pTRAC-373-1706 expression vector.

The large arrows represent the open reading frames of β-lactamase (bla),the thermosensitive repressor cl⁸⁵⁷ of phage lambda (λcl⁸⁵⁷), the cyaCgene and truncated 'cyaA gene (the arrows are pointing to the directionof translation of the corresponding genes). The ColE1 origin (Ori), thePr promoter (λPr), and some relevant restriction sites are alsoindicated. The intergenic region between the cyaC and truncated 'cyaAgenes is detailed in the lower part. It shows the new C-terminusextension of cyaC (downstream to Arg182 of wild-type cyaC), the stopcodon (underlined), the initiator Met and first codons of CyaA373-1706(upstream to Ser373 of wild-type CyaA).

EXAMPLES A. The Bordetella Adenylate Cyclase Toxin InteractsSpecifically with the α_(M)β₂ Integrin (CD11b/CD18)

A.1 Materials and Methods

A.1.1 Recombinant Toxins and Antibodies

Protocol for CyaA production has already been described elsewhere[Karimova, et al, 1998]. CyaA toxins were produced in E. coli BLR strainharboring an expression plasmid, pCACT3, which carries the cyaAstructural gene CyaA under the lacUV5 promoter and the cyaC accessorygene required for activation of the protoxin. After solubilization in 8Murea, Hepes-Na 20 mM, pH 7.5, CyaA was purified to more than 95%homogeneity (as judged by SDS-gel analysis, not shown) by sequentialDEAE-Sepharose and Phenyl-Sepharose. A recombinant detoxified CyaAtoxin, CACTE5-Cys-Ova, harboring a unique cysteine inserted within thegenetically inactivated catalytic domain was constructed by inserting anappropriate double strand oligonucleotide between the BsiwI and KpnIsites of pCACT-Ova-E5 [Guermonprez et al, 2000]. In the resultingprotein CACTE5-Cys-Ova, the amino acid sequence ASCGSIINFEKLGT isinserted between residues 224 and 225 of CyaA. The recombinant toxin wasexpressed and purified as previously described. The purified protein waslabeled on its unique Cys with the highly specific sulfhydryl reagentN-(6-(Biotinamido)hexyl))-3′-(2′-pyridyldithio) propionamide(Biotin-HPDP, PIERCE) according to the manufacturer's instructions. Thebiotinylated-CyaA was repurified on DEAE-Sepharose to remove theunreacted Biotin-HPDP reagent. Toxin concentrations were determinedspectrophotometrically from the adsorption at 280 nm using a molecularextinction coefficient of 142 M⁻¹×cm⁻¹ (binding studies) or using theBiorad protein assay system (cAMP accumulation and cell death studies).Purified mAbs specific for murine CD11a (2D7, Rat IgG2a, κ), murine andhuman CD11b (M1/70, Rat IgG2b, κ), murine CD11c (HL3, Hamster 1, λ),murine CD18 (C71/16, Rat IgG2a, κ), control (A95-1, or anti-CD16/32,2.4G2, Rat IgG2b, κ) originated from Pharmingen (San Diego, USA).Supernatants from anti-human CD11b (44, Mouse IgG2a, κ), and anti-humanCD18 (TS/18, Mouse IgG1, κ) hybridoma were a kind gift and were used at½ dilution in blocking experiments. Supernatents from an anti-murineCD11b (5C6, Rat IgG2b, κ) were a kind gift from G. Milon (PasteurInstitute, Paris) and were used at ½ final dilution in bindinginhibition assays. Anti-CyaA polyclonal antibodies were obtained from arabbit immunized subcutaneously with purified CyaA. Sera wereprecipitated from immune serum by ammonium sulfate (33%). Aftercentrifugation the pelleted proteins were resuspended in 20 mM Hepes-Na,150 mM NaCl, pH 7.5 (buffer C) and extensively dialysed against the samebuffer. The antibodies were then biotinylated by incubation withBiotin-amidocaproate N-Hydroxysuccinimide ester (SIGMA, dissolved indimethyl sulfoxide) for 130 min at room temperature. Then, 100 mMethanolamine, pH 9.0 were added and after 30 min of additionalincubation, the mixture was extensively dialysed at 4° C. against bufferC. Biotinylated antibodies were stored at −20° C.

A.1.2 Cells and Culture Media

EL4, J774A.1, LB27.4, THP-1 were obtained from the American Type CultureCollection (ATCC) and were cultured in RPMI 1640 supplemented with 10%fetal calf serum, 100 U/ml penicillin, 100 μg/ml streptomycin, 2mML-glutamine, with or without 5×10⁻⁵ M 2-mercaptoethanol (completemedium). FSDC [Girolomoni et al, 1995] were cultured in complete medium.CHO cells transfected for human CD11b/CD18 or CD11c/CD18 or transfectedwith the vector only were obtained from D. Golenbock (Boston, USA) andcultured in the presence of neomycin as previously described [Ingalls etal, 1998]. Human neutrophils were purified as previously described [Rieuet al, 1992].

A. 1.3 CyaA Binding Assays

All binding assays were performed in DMEM 4.5 mg/ml glucose (LifeTechnologies) without serum in 96 well culture plates (Costar). 2×10⁵cells/well were incubated for 20 minutes (at 4° C. or 37° C. dependingon the experiments) in a 200 μl final volume. In some experiments, cellswere preincubated for 20 min at 4° C. in the presence of blocking mAbsin 100 μl final volume. The toxin solution was added to the wells in thecontinuous presence of the mAbs in a total volume of 200 μl at 4° C.Then, plates were centrifuged at 1500 rpm for 5 min and supernatentswere removed. Cells were incubated at 4° C. for 25 minutes withbiotinylated anti-CyaA rabbit polyclonal antibodies (1/400 in DMEM, 50μl/well) in the presence of a control (non-immune or pre-immune) rabbitserum as a saturating agent (1/50).

After centrifugation and supematant removal, cells were stained withstreptavidin-phycoerythrin (PE) (Pharmingen) at 1/300 dilution (50μl/well). After washing, cells were analyzed by flow cytometry on aFACStar (Becton-Dickinson, Mountain View, USA) in the presence of 5μg/ml propidium iodide. Gatings were done to exclude cells aggregatesand dead cells by propidium iodide exclusion. Experimental points werefitted to a hyperbolic model ΔMFI=Bmax*[CyaA]/(K_(d)+[CyaA]), withBmax=% of maximal binding, using the prism software.

A.1.4 cAMP Assay

cAMP accumulation was measured by an antigen competition immunoassay[Karimova et al, 1998] in which the incubation medium was composed ofDMEM without serum but containing 4.5 mg/ml glucose and 20 U/mlhexokinase. Hexokinase, which catalyzes the ATP-dependentphosphorylation of glucose, was added deplete the extracellular mediumfor any traces of ATP, thus preventing the extracellular synthesis ofcAMP. Therefore, the amount of cAMP measured is representative of theaccumulation of strictly intracellular cAMP.

5×10 ⁵ cells were preincubated in 96 well plates in 100 μl/well with orwithout 10 μg/ml of specific mAbs at 4° C. for 1 h and then incubated at37° C. for 20 min with 0.05, 0.5 or 5 g/ml CyaA and with 10 μg/ml ofspecific mAbs when present during the preincubation. For the doseresponse effect of CyaA, cells were directly incubated with the toxinfor 20 min at 37° C. After intoxication, cells were centrifuged at 2.500rpm for 5 min. Samples were lysed with 100 μl of HCl 0.1 N, boiled for 5min at 120° C., and neutralized with 100 μl of Tris 0.125 M, NaCl 0.2 M.Microtiterplates were coated with cAMP-BSA conjugates diluted at 1/4.000in Na₂CO₃ 0.1 M pH 9.5. They were washed twice in PBS-Tween 0.1%,saturated for 1 h in PBS-BSA 2% and washed five times with PBS-Tween0.1%. The samples and the cAMP standard (Sigma) were directly added tothe plates coated with cAMP-BSA conjugates and serially diluted with a1/1 mixture of HCl 0.1 N and Tris 0.125 M-NaCl 0.2 M. Anti-cAMP rabbitantibody was added at 1/2.500 in PBS-BSA 2% and incubated at 37° C. for3 h. Plates were washed five times with PBS-Tween 0.1%. Anti-rabbitantibodies coupled to horseradish peroxidase (Amersham) were added at1/2.500 in PBS-BSA 2%, incubated at 37° C. for 1 h and revealed usingthe classical peroxidase reaction. Experimental points of the standardcurve were fitted to a sigmoid model using the Prism software.

A.1.5 Toxicity Assay

Cell death was evaluated as previously described [Khelef et al, 1993;Khelef et al, 1995]. Briefly, 10⁵ cells were incubated for 24 hours in a96 well plate in complete medium and washed once with serum free medium.All cell incubations were further performed in serum free medium. Doseresponse effects were evaluated by directly applying variousconcentrations of CyaA to CHO cells at 37° C. for 4 h. For cytotoxicityinhibition, cells were preincubated at 4° C. for 1 h with or without 10μg/ml of specific mAbs and then incubated at 37° C. with 0.5 μg/ml CyaAfor 2 h for J774A.1A.1 cells or with 5 μ/ml for 4 h for CHO cells andwith 10 μg/ml of specific mAbs when present during the preincubation.Cell lysis was evaluated using the Cytotox 96™ assay (Promega) whichquantifies the amount of lactate dehydrogenase (LDH) released in themedium by dying cells.

A.2 Results

A.2.1 Saturable Binding of CyaA Correlates with the Presence of CD11b atthe Surface of Target Cells

To characterize CyaA cellular specificity toward a population ofleukocytes, we choose three representative murine cell lines expressingvarious combination of β₂ integrins: J774A.1, a tumoral macrophage; EL4,a T cell thymoma, and LB27.4, a B cell lymphoma. After a 20 minutes ofincubation with CyaA at 37° C., binding of CyaA to the cell surface ofthese cells was monitored by flow cytometry using biotinylated anti-CyaAantibodies and streptavidine-PE. Under these conditions, we observed anefficient, dose-dependent and saturable binding of CyaA on J774A.1 cellline. The affinity of CyaA for J774A.1 cells was high since the apparentK_(d) were 9.2±4.5 nM and 3.2±1.9 nM, respectively. A low binding ofCyaA to EL4 and LB27.4 cells was observed but it was not saturable atthe concentrations tested.

In order to determine if the binding of CyaA to J774 cell lines wascorrelated to the expression of one of the members of the β₂ integrinfamily, we performed a phenotypic analysis of these cells by flowcytometry using monoclonal antibodies (mAbs) specific for the three achains of the well characterized β₂ integrins (CD11a, Cd11b and CD11c)and for the common β chain (CD18) (FIG. 1 b, c, d, e). J774A.1 cellsexpressed mostly CD11b and CD18, but were also positive for CD11a. EL4cells and LB27.4 cells expressed mostly CD11a and CD18. Taken together,these data show that the efficient and saturable binding of CyaA toJ774A.1 was correlated with the presence of the integrin CD11b/CD18.

-   49. The proteinaceous vector according to any one of claim 47 or 48    wherein the adenylcydase is a non toxic adenylcydase.-   50. The proteinaceous vector according to any one of claims 47 to    49, wherein the genetically modified adenylcyclase is a Bordetella    adenylclase lacking all or part of its N-terminal catalytic domain.-   51. The proteinaceous vector according to any one of claims 43 to 50    wherein the adenylcyclase is from Bordetella pertussis.-   52. The proteinaceous vector according to claim 51, wherein said    recombinant adenylcyclase is the Bordetella pertussis adenylcyclase    lacking residues 1-373.-   53. The proteinaceous vector according to claims 43 to 52, wherein    said molecule of interest is selected in the group comprising:    peptides, glycopeptides, lipopeptides, polysaccharides,    oligosaccharides, nucleic acids, lipids and chemicals.-   54. The proteinaceous vector according to any one of claims 43 to    53, wherein said molecule of interest is a drug chemically or    genetically coupled to said adenylcyclase.-   55. The proteinaceous vector according to claim 54, wherein the    molecule of interest is an epitope.-   56. The proteinaceous vector according to any one of claim 54 or 55,    wherein said molecule of interest is an epitope chemically coupled    to a genetically modified adenylcyclase devoid of native cysteine    residue but containing genetically inserted cysteine residue(s) at    permissive site(s) within its catalytic domain.    A.2.2 CyaA Saturable Binding is Specifically Blocked by anti-CD11b    mAbs

We next examined if CD11b /CD18 could be directly involved in thebinding of CyaA to the cells expressing this integrin. We performed aquantitative analysis of inhibition obtained with anti-CD11b M1/70 mAbby calculating percentage of mean fluorescence values in the absence ofmAbs at a fixed or varying concentrations of CyaA (FIG. 2). Inhibitionof CyaA binding obtained with the M1/70 anti-CD11b mAb was almost totalat most CyaA concentrations tested (FIG. 2 a, b): This inhibition wasspecific since anti-CD11a, CD11c, CD18 or a control mAb did not inhibitCyaA binding. A second anti-CD11b mAb (clone 5c6) also inhibited bindingof CyaA (FIG. 2 c, d). Similar results were obtained with FSDC, animmature dendritic cell line expressing CD11b (FIG. 2 a, c), and theJ774A.1 macrophage cell line (FIG. 2 b, d).

To examine whether CyaA could similarly interact with human CD11b, CyaAbinding studies were performed on human neutrophils, whose highexpression of CD11b is well established. Since high backgroundfluorescence was obtained following incubation of human myeloid cellswith the anti-CyaA rabbit antibodies (data not shown), we set up analternate binding assay. A detoxified form of CyaA was specificallybiotinylated on unique cysteine residues, genetically introduced withinits catalytic domain. Using this system, we were able to detect CyaAbinding to neutrophils (FIG. 3). Preincubation of neutrophils with the44 or M1/70 anti-CD 11b mAbs led to, a respectively complete or partialinhibition of the binding of CyaA (FIG. 3 a and b). Unlike the C71/16,anti-murine CD18 mAb that did not block CyaA binding to murine cells,preincubation with the human anti-CD18 TS/18 mAb led to a completeinhibition of CyaA binding to human neutrophils (FIG. 3 b). Similarresults were obtained with the human monocytic THP-1 cell line (data notshown).

In conclusion, CyaA binding to the surface of three myeloid cell linesfrom both murine and human origin (J774A.1, FSDC, THP-1) as well asfreshly purified human neutrophils appears to be mainly mediated throughthe CD11b/CD18 integrin.

A.2.3 CyaA-media ted cAMP Increase and Toxicity are Specifically Blockedby an anti-CD11b mAb

To evaluate the physiological relevance of CD11b/CD18-dependent CyaAbinding, we studied the effect of blocking mAbs on the cytotoxicity ofCyaA. We first measured the amount of cAMP produced in J774A.1 cellsexposed to CyaA in the presence of various mAbs. As shown in FIG. 4 a,the increase in the intracellular cAMP content induced by CyaA wastotally abolished when cells were preincubated with the M1/70 anti-CD11bmAb. The C17/16 anti-murine CD18 mAb had that did not block CyaA cellbinding (FIG. 2) had no effect on the intracellular cAMP content of CyaAtreated cells. Thus, these data strongly suggest that the increase inintracellular cAMP induced by CyaA is dependent upon the interaction ofthe toxin with CD11b . To further analyze the requirement of thismolecule for the toxicity of CyaA, we evaluated the effect of mabsspecific for the different chains of the β₂ integrin family on CyaAmediated cell death. FIG. 4 b shows that anti-CD11b mAb J774A.1dramatically reduced the cell death induced by CyaA (88% inhibition).The cell death induced by CyaA was unaffected when J774A.1 werepreincubated with mAbs that did not block toxin binding to cells(anti-CD11a, CD11c or CD18 or a control mAb).

Taken together, these data indicate that CyaA binding through CD11b isstrictly required for CyaA mediated toxicity in J774A.1 cells.

A.2.4 Transfection of CHO cells with CD11b/CD18 Confers Sensitivity toCyaA

To confirm the role of CD11b in CyaA binding, we used CHO cellstransfected with the human integrins CD11b/CD18 or CD11c/CD18 or mocktransfected (vector alone). As shown in FIG. 5 a, CyaA could bind, at37° C., to these cell lines. However, CyaA binding was efficient andsaturable in CHO cells expressing CD11b/CD18 but not in CD11c/CD18 ormock-transfected cells. The affinity of CyaA for CD11b/CD18 transfectedcells was in the nM range (Kd=0.7±0.09 nM). At 4° C., the efficiency ofCyaA binding was reduced as compared with the binding at 37° C. (FIG. 5b). At this temperature, the differences between CD11b/CD18 transfectedcells and the two other cell lines were more pronounced.

Since we found that CD11b was required for CyaA mediated toxicity inJ774A.1, we then determined if CD11b expression was sufficient to confera CyaA-sensitive phenotype to CHO transfected cells. In line withprevious reports [Gordon et al, 1988], CyaA induced a notable amount ofintracellular cAMP in CHO cells transfected with CD11c/CD18 or incontrol mock-transfected cells, but only at high concentrations of toxin(5 μg/ml, FIG. 5 c). In contrast, CyaA increased intracellular level ofcAMP in CD11b/CD18 transfected CHO cells even at the lowestconcentration studied (0.05 μg/ml). Moreover, the cAMP production inresponse to 5 μg/ml CyaA was 4 to 5 times more elevated in CD11b/CD18transfected cells as compared to CD11c/CD18- or mock-tranfected cells.

We also evaluated the role of CD11b/CD18 in CyaA-mediated cell death. Asshown in FIG. 5 d, more than 50% of CHO transfected with CD11b/CD18 werekilled after 4 hours incubation with 5 μg/ml of CyaA, whereasCD11c/CD18- or mock-transfected cells were not affected by thistreatment.

Altogether, these results thus clearly established that expression ofhuman CD11b /CD18 integrin is sufficient to create a high affinityreceptor for CyaA in CHO cells.

A.3 Discussion: A Receptor for CyaA

Unlike other toxins, CyaA has been considered for a long time, asindependent of any receptor binding. This is based on the observationsthat i) CyaA can intoxicate in vitro a wide variety of model cell linesfrom various origin [Ladant et al, 1999] ii) CyaA binds to Jurkat cellsand sheep erythrocytes in a non saturable fashion [Gray, et al 1999]. Infact, these observations established that non-specific adsorption ofCyaA to lipid membranes leads to some translocation of the catalyticdomain into the cytosol. However, they did not rule out the existence ofa specific receptor. We showed in this study on myeloid cell lines thatthe binding and the toxic properties of CyaA are dependent on itsinteraction with the integrin CD11b/CD18. Efficient and saturablebinding correlates with the expression of CD11b and is fully andspecifically blocked by anti-CD11b mAbs. Moreover, expression ofCD11b/CD18 in CHO cells dramatically enhances the binding of CyaA,resulting in an increased sensitivity to intoxication by this toxin. Ourresults are the first evidence supporting the interaction of CyaA with acell-surface molecule specifically expressed on leukocytes. The nearlycomplete blockade of CyaA binding by anti-CD11b mAbs suggests that CD11bis the main receptor for CyaA in the cell lines tested. The lack ofefficient binding to CD11c/CD18 transfectants, or CD11a/CD18 expressingcells such as EL4 or LB27.4 suggest that CD11b/CD18 is the only integrinof the β2 family involved in the binding of CyaA to target cells.

In line with previous studies, we observed a detectable binding of CyaAto all cell lines tested. Furthermore, CyaA at high concentrationstriggered a small but detectable cAMP increase in mock-transfected CHOcells, that is not associated to cell death. Thus, at highconcentration, CyaA can bind and translocate to a wide variety of celllines but efficient and saturable binding, translocation and killing isa hallmark of CD11b expressing cells.

Binding of CyaA to a member of the β2 integrin family is reminiscent ofthe behavior of other RTX toxins which were recently found to interactwith these molecules [Lally et al, 1997; Li et al, 1999; Ambagala et al,1999; Jeyaseelan et al, 2000]. The E. coli HlyA, that shares a stronghomology with CyaA, forms cationic pores at the plasma membrane. HlyAexhibits a specificity for leukocytes but only at low concentration[Welch et al, 1991]. This relative specificity was shown to be mediatedby its interaction with the integrin CD11a/CD18 [Lally et al, 1997].Similarly, A. actinomycetemcomitans and P. haemolytica leukotoxin (LtxAand LktA, respectively), which are less promiscuous RTX toxins specificfor human and bovine leukocytes, respectively, also interact withCD11a/CD18 [Lally et al, 1997; Li et al, 1999; Ambagala et al, 1999;Jeyaseelan et al, 2000]. Despite its strong homology with HlyA, CyaArecognizes another β₂ integrin (CD11b/CD18) whose cellular distributionis different. Indeed, CD11b is expressed mostly on macrophages,neutrophils and dendritic cells, but not on the majority of T and Bcells, whereas CD11a is expressed on all leukocytes including T and Blymphocytes.

This specific targeting of CyaA to CD11b expressing cells is exploitedin the present invention to specifically target this particular subsetof cells. Detoxified mutants of CyaA remaining invasive could be usedfor the delivery of pharmacologically active molecules to CD11b positivecells, without noticeably affecting other cell types.

B. Targeted Antigen Delivery to the Cytosol of Myeloid Dendritic Cellsand Selective CTL Priming

B.1 Materials and Methods

B.1.1 Recombinant Adenviate Cyclase Toxins and Petide

The pOVA synthetique peptide (SIINFEKL) originated from NEOSYSTEM andwas diluted in PBS at 1 mg/ml.

B.1.2 Immunization and Assay for the Detection of Cytotoxic T Cells

Female C57BL/6 (H-2^(b)) from Iffa Credo (L'arbresle, France) were usedbetween 6 and 8 weeks of age. TAP1−/− (Van Kaer et al., 1992), CD4−/−(Killeen et al., 1993), CD40−/− (Kawabe et al, 1994) and B celldeficient μMT (Kitamura et al, 1991) bred onto a C57BL/6 backgroundoriginated from the CDTA facility (CNRS, Orleans, France) and were bredin the Institut Pasteur facilities. Animals were intravenously immunizedwith Ag in PBS. Seven days post-injection, animals were sacrified andthe spleen was removed. Single cell suspensions of splenocytes (2.5×10⁷cells) were restimulated in 10 ml CM (see below) with irradiated spleencells (2.5×10⁷ cells) for 5 days in the presence of 1 μg/ml pOVA.Cytotoxicity assay was performed exactly as previously described(Fayolle, et al., 1999).

B. 1.3 Cell lines

B3Z (Karttunen et al., 1992), a CD8+ T cell hybridoma specific for theOVA 257-264 peptide (SIINFEKL) in the context of H-2K^(b) was a generousgift from Dr N. Shastri (University of California, Berkeley, USA).

B.1.4 Antigen Presentation Assays

All antigen presentation assays were performed by coculture of APC withB3Z in 96 well culture microplates (0.2 ml final volume) in RPMI 1640supplemented with 10% Fetal Calf Serum, 100 U/ml penicillin, 100 μg/mlstreptomycin, 2 mM L-glutamine, and 5×10⁻⁵ M 2-mercaptoethanol (completemedium, CM). The stimulation of B3Z cells (10⁵ cells/well) was monitoredby IL-2 release in the supernatents of 18-24 hours cultures in thepresence of APC. IL-2 was measured in CTLL assay as previously described(Guermonprez et al., 1999). In some experiments (see figure legends),B3Z stimulation was assessed using the NF-AT lacZ reporter assay. LacZactivity in cell lysates was assessed with the CPRG substrate aspreviously described (Karttunen et al., 1992). Two antigen presentationassays were performed: i) In vitro assay: APC originated from naive micewere cocultured (10⁵/well) with B3Z in the presence of Ag at variousconcentrations. In some experiments, APC were preincubated or not withmAbs at 10 μg/ml for 40 minutes at 4° C., then Ag was added to the cellsin a 100 μl final volume in the continuous presence of the mAbs. After a4 hours pulse, APC were washed twice and put in coculture with B3Z.Purified mnAbs used were against CD11b (M1/70 ratigG2b, κ) orisotype-matched control and originated from Pharmingen (San Diego, USA).ii) Ex vivo assay: APC originated from mice previously intravenouslyimmunized with various Ags and were put in culture with B3Z in a 0.2 mlfinal volume at various numbers of APC per well.

B.1.5 Antigen Presenting Cells and Sortings

Total spleen cells (TSC), low density (LDF) and high density (HDF)fractions were prepared according to the protocole of Steinman modifiedby Vremec et al. (Vremec et al., 1992). Briefly, spleens were digestedwith collagenase for 40 minutes at 37° C. and then dilacerated andprepared in the continuous presence of EDTA 5 mM. Cells werecentrifugated on a dense BSA solution. Supematant and pellet cells werecollected appart and termed low density and high density fractions.CD11c staining was performed at 4° C. in PBS supplemented with 5% ofFetal Calf Serum and 2 mM EDTA (PBS-FACS) with the hamster HL3 mAbcoupled to phycoerythrin (PE), fluoresceine isothyocyanate (FITC) orbiotinylated and then revealed by Streptavidine-PE. CD8α staining wasperformed with the 53-6.7 mAb coupled to PE. CD11b staining wasperformed with the M1/70 mAbs coupled to PE or FITC. CD54R staining wasperformed with the B220 mAb coupled to PE or biotinylated and revealedby streptavidine-PE. All mAbs originated from Pharmingen. After twowashes, cells were sorted using a FACStar (Beckton Dickinson, MountainView, USA). Cells were aseptically recovered in CM. Purity of the sortedcells was checked on an aliquot of the sorted cells analyzed on aFACScan apparatus (Beckton Dickinson, Mountain View, USA). Purity of thesorted cells was typically between 80 and 98%. In other experiments (asmentioned in figure legends), CD11c+ cells were sorted directly fromspleen collagenase digests using the CD11c Micro Beads and the MagneticCell Sorting technology following the supplier protocole (MACS,MiltenyiBiotec, Bergish Gladbach, Germany). Purity of the sorted cellsranged around 80% with this technique.

B.2 Results

B.2.1 CD4- and CD40-Independent CTL Primina after Systemic Immunizationwith CyaAOVA in the Absence of Adjuvant

The chicken ovalbumin, H-2K^(b) restricted, SIINFEKL epitope was used asan experimental model epitope. It was genetically inserted in thecatalytic domain of a detoxified, still invasive mutant CyaA. C57BL/6(H-2^(b)) mice were immunized iv once with 50 μg of the recombinanttoxin or control saline solution. Seven days after immunization, CTLactivity specific for pOVA was detected within splenocytes ofCyaAOVA-immunized C57BL/6 mice but not in mice injected with saline or acontrol CyaA (FIG. 6 a). Similar results were obtained with CD4 or CD40deficient mice indicating that, unlike many other CTL responses (asthose raised against cross-priming Ag (Bennett et al, 1997; 1998;Schoenberger et al., 1998, Ridge et al., 1998) CD4+ T cell help was notmandatory for priming of CTL responses by CyaAOVA (FIG. 6 b, c).Furthermore, B cells were not required since CTL responses were alsoobtained in B cell deficient mice (IgM−/−, FIG. 6 d). Contaminant LPS,possibly acting as adjuvants, are not involved in the stimulation of CTLresponses CyaAOVA since C57BL10ScSn and the LPS-hyporesponsive miceC57BL10ScCr displayed similar OVA-specific CTL response after CyaAOVAinjection (not shown).

B.2.2 gn Vitro and in Vivo targeting of CyaAOVA Presentation toCD11b-Expressing Cells

In order to better understand the immunogenicity of CyaAOVA, we intendedto determine the APC involved in its presentation to CD8+ T cells. Usingthe IL-2 secretion as a readout for stimulation, we show that B3Z, anH-2K^(b)-restricted, anti-OVA CD8+ T cell hybridoma, is stimulated invitro by bulk splenocytes in the presence of CyaAOVA but not CyaALCMV(FIG. 7 a). We intended to analyze the APC ability of three well definedsplenic APC: DC (CD11c+), B cells (CD45R⁺) and macrophages/granulocytes(CD11b^(high)+CD11c−). Due to the low percentage of CD11c+ in totalsplenocytes (<1%), we perforrned density fractionation and sorted theCD11c+ from the DC-enriched (4-10%) low density population by flowcytometry. The high density fraction contained only trace amounts ofCD11c⁺ cells. In contrast, the distribution of CD45R+ andCD11b^(high+)CD11c⁻ (composed of granulocytes/macrophages) cells in bothfractions allowed sortings on the total population for these markers. Asshown in FIG. 7 c, CyaAOVA was presented most efficiently by DC, lessefficiently by CD11b^(high+)CD11c⁻ and B cells. This correlates with aquasi exclusive distribution of antigen presenting ability within thelow density fraction of splenocytes (FIG. 7 a). In sharp contrast, bothhigh and low density fractions were able to stimulate B3Z in response topOVA (not shown).

To detect K^(b)-OVA complexes formed in vivo after immunization, the APCprepared from mice iv immunized 8-15 hrs before with 50 μg CyaAOVA werecocultured with B3Z in vitro without Ag addition (ex vivo assay). As forin vitro assays, APC responsible for CyaAOVA presentation were presentexclusively in the DC-enriched low density fraction of splenocytes (FIG.7 b). The specificity of the assay was checked with APC fromCyaALCMV-immunized mice that did not stimulated B3Z (FIG. 7 b). In orderto further characterize the low density APC involved in CyaAOVApresentation, we performed call sorings: results shown in FIG. 7 d showthat DC (CD11c⁺) fraction was the more efficient APC. Themacrophage/granulocyte (CD11b^(high+)CD11c⁻) and the B cells (CD45R⁺)sorted from the same fraction were very inefficient for the stimulationof B3Z

To further characterize APC involved in CyaAOVA, we performedsubfractionation of the splenic low density CD11c⁺DC in CD11c⁺CD8α⁻myeloid subset and CD11c⁺CD8α⁺ lymphoid subset. In vitro and ex vivoassays (FIG. 7 e and f, respectively) showed that antigen presentingability for CyaAOVA was retained by the myeloid subset that alsoexpressed CD11b⁺, unlike the lymphoid subset expressing low levels ofthis integrin (Pulendran et al., 1997; Vremec et al., 1997). Theinability of the CD11c⁺CD8α⁺ lymphoid subset to present CyaAOVA wasspecific for CyaAOVA since CD11c⁺CD8α⁺ and CD11c⁺CD8α⁻ presented equallywell the pOVA synthetic peptide to B3Z.

In vitro and ex vivo assays performed with splenocytes from control(C57BL/6) or B cell-deficient mice (IgM−/−) confirmed the poorcontribution of B cells to CyaAOVA presentation in vitro and in vivo(FIG. 7 g and h). In line with these results, CTL responses induced byCyaAOVA were not significantly affected in B cell-deficient mice ascompared to control mice (FIG. 5 d).

B.2.3 MHCl-restricted Presentation of CyaAOVA by Dendritic Cells Dependson the Cytosolic Delivery of the Recombinant Toxin Both in Vitro and inVivo

To determine whether CyaAOVA presentation depends on the cytosolicdelivery of the OVA epitope or extracellular loading, we performed Agpresentation assays with total splenocytes or CD11 c⁺ DC from TAP−/− orcontrol TAP+/+ splenic purified from naive animals (in vitro, FIG. 8 a)or from animals immunized iv with CyaAOVA (ex vivo, FIG. 8 b). Resultsshow that CyaAOVA presentation by dendritic cells is fully dependent onTAP either in vitro or in vivo. Since pOVA presentation is aTAP-independent phenomenon, we checked the functionality of TAP−/− DCsorted from CyaAOVA-immunized mice by stimulating B3Z with these cellsloaded in vitro with the pOVA peptide (not shown). These results showthat in vivo presentation of CyaAOVA depends on its cytosolic delivery.

B.2.4 Interaction of CyaA with CD11b is Required for Cell Binding andDelivery of the Inserted Antigen to the Cytosolic Pathway for AntigenPresentation by MHCl

We showed on part A that saturable and efficient binding of CyaA WT toCD11b⁺ cells can be blocked specifically by anti-CD11b mAbs. Moreover,CD11b transfection specifically confered saturable binding andsensitivity to CyaA WT to otherwise CD11b⁻ cells resistant to CyaA WT.Cell binding blockade by anti-CD11b mAbs inhibited subsequentintracellular delivery of the catalytic adenylate cyclase domain, cAMPelevation and cell death induced by CyaA WT. Since this results havebeen obtained on cell lines, it remained to determine if CyaA binds tosplenocytes. We set up a flow cytometric assay to detect the fixation ofa biotinylated, detoxified form of CyaA carrying the OVA peptide(CyaAOVAbiotine) to total splenocyte suspensions with streptavidincoupled to phycoerythrin. Using this assay, we observed that CyaAOVAbinds to a subset of leukocytes within the total splenocyte suspension(5-7%). Preincubation with the anti-CD 11b M1/70 mAb but not a controlmAb abrogated this binding (FIG. 9 a). Furthermore, there is acorrelation between the expression of CD11b and CyaAOVA binding to lowdensity cells: CyaAOVA binds efficiently to CD11c⁺CD8α⁻ that expresseshigh levels of CD11b, less efficiently to CD11c³⁰CD8α⁺ that expresseslow levels of CD11b and very weakly to CD11c⁻CD8 α⁺ T cells that do notexpress CD11b (FIG. 9 b). It is noticeable that CyaOVA binds efficientlyto a low percentage of CD11c⁻CD8α⁻ cells in correlation with thepresence of CD11b ^(high+) into this CD11c⁻ population. Thus, CyaOVAbiotine binding is mediated by CD11b (as for CyaA WT) and predicts theability of a given cell type to present CyaOVA.

In vitro, we showed that the anti-CD11b mAb M1/70 blocks the CyaAOVApresentation by TSC cells to B3Z (FIG. 10 a). This blockade is specificsince i) a control mAb or mAbs specific for other β2 integrin familymembers (anti-CD11a, CD11c) had little or no effect (FIG. 10 a and datanot shown) ii) the presentation of pOVA was not affected by theanti-CD11b or none of these mAbs (FIG. 10 b and data not shown). Thisconfirms that CD11b is the main receptor for CyaAOVA at least in thespleen, and that CyaAOVA-CD11b interaction is mandatory for thepresentation of the inserted epitope. Finally, to ascertain the role ofCD11b expressing cells in CyaAOVA presentation, we performed sortingexperiments on whole splenocyte cells from mice immunized with CyaAOVAor pOVA. Total splenocyte cells were sorted in CD11b⁺ and CD11b⁻fractions. Whereas the two subpopulations stimulated B3Z after pOVA ivimmunization (FIG. 10 d), only the CD11b⁺ subpopulation stimulated B3Zafter CyaAOVA iv immunization (FIG. 10 c).

Taken together, these results clearly establish that the presentation ofthe OVA peptide from CyaOVA is dependent on cell binding and thus oninteraction with CD11b.

B.3 Discussion

In the present study, using the detoxified adenylate cyclase ofBordetella pertussis as an epitope-delivery vector, we established astrategy for immunization that primes CTL responses after a singleinjection, bypassing the need for adjuvant requirement. We identifiedmechanisms that contribute to the high efficiency of detoxified CyaA asa vector:

B.3.1 CyaA Target Myeloid Dendritic Cells Through its Interaction withthe CD11b Integrin

Antigen presenting assay to a specific CD8+ T cell hybridoma using invitro or in vivoloaded APC (in vitro and ex vivo assay, respectively)demonstrated that the most efficient APC for CyaAOVA areCD11c+CD11b^(high+) DC. Indeed, all the Ag presenting ability forCyaAOVA belonged to the low density fraction of splenocytes that retainsDC. Cell sorting of defined cell types revealed thatCD11c³⁰CD11b^(high+) DC cells are much more efficient thanCD11c⁻CD11b^(high+) cells to present CyAOVk The minor contributionobserved for B cells (CD45R⁺) was confirmed by efficient presentation ofCyaAOVA (in vitro and ex vivo) and CTL responses in B cell-deficientmice.

B.3.2 CyaA Delivers Ag to the Cytosolic Pathway for MHC class IPresentation in Vivo

Here we show the dependence of CyaAOVA presentation to total splenocytesin vitro. Strikingly, we also show that in vivo presentation of CyaAOVAtakes also place according a TAP-dependent pathway. This lead to theconclusion that CyaAOVA presentation in vivo resulted effectively fromcytosolic delivery and not from an eventual extracellular degradation.

B.3.3 CTL Priming Bypass CD4+ T cell help and is independent on CD40Signaling

Maturation from an immature stage toward a mature stage is characterizedby i) a decrease in Ag capture ability, ii) an increase in T cellpriming ability, iii) a migration from Ag sampling sites (marginal zonein the spleen) toward T cell area (peri arteriolar sheets in the spleen)were they maximize the probability of encounter with Ag-specific T cells(De smedt et al., 1996). In addition to Ag presentation by DC, thematuration phase is now widely assumed as a prerequisite for T cellpriming. In vitro) studies have highlighted the role of CD4⁺ T cells insignalling DC maturation, notably via CD40L-CD40 interaction (Bell etal., 1999). In the case of CD8+ T cells priming after the crossprimingof cellular Ag, CD4⁺ T cells dispensate their help to CD8+ T cells in aCD40-dependent mechanism (Schuurhuis et al., 2000; Bennett et al., 1998;Schoenberger et al., 1998; Ridge et al., 1998). Since CyaAOVA primes CTLin a CD4 and CD40 independent way, it is tempting to speculate thatdetoxified CyaA could be endowed of intrinsic adjuvanucity.

B.3.4 Conclusion

The present study represents the first characterization, to ourknowledge, of a proteinaceous vaccine vector that fits both targeting toprofessional APC, cytosolic delivery of the vectorized Ag andadjuvant-free CTL priming. Moreover, we elucidated the mechanism of celltargeting by demonstrating that Ag presentation is dependent on theinteraction between CyaA and CD11b, its receptor. Hence, the cellularspecificity of CyaA is serendipituously adapted to the Ag deliverypurpose. Finally, the cellular specificity of CyaA or other bacterialtoxins may serve the cytosolic delivery of a wide set ofpharmaceutically-relevant molecules whose effects should be targeted ona restricted set of cells.

C. Use of adenylcyclase to Deliver Chemically Coupled Antigens toDendritic Cells In Vivo

C.1 Methods for Coupling Molecules of Interest to CyaA-derived Vectors

A general methodology is here described to create recombinant CyaAtoxins by grafting molecules of interest to CyaA by means of disulfidebonds.

As an illustration, a synthetic 12 amino-acid long peptide correspondingto a CD8⁺ T-cell epitope from ovalbumin was chemically crosslinkedthrough a disulfide bond to a cysteine residue genetically introducedinto the CyaA catalytic domain at position 235 (wild type CyaA has nocysteine residues). The expected advantages of this novel architectureare:

-   -   (i) versatility: a single CyaA carrier protein can be easily        coupled to any desired synthetic peptides;    -   (ii) immunogenicity: upon delivery into the APC cytosol, the        epitopes chemically coupled to CyaA should be released from the        vector (due to the reducing intracellular conditions) and        introduced directly into the MHC class I presentation pathway,        thus bypassing the potentially limiting step of proteolytic        processing by proteasome.

The general procedure to couple synthetic peptides to CyaA by disulfidebonds is outlined in FIG. 11.

In a first step, a recombinant CyaA toxin that contains a singlecysteine residue genetically inserted within the catalytic domain ofCyaA (wild type CyaA has no cysteine residue) is produced.

The recombinant CyaA toxin, ACTM235, has been previously characterized(Heveker and Ladant, 1997). In particular, ACTM235 harbors a Cys-Serdipeptide inserted between amino-acid 235 and 236 and is fullycytotoxic. This toxin was expressed and purified to homogeneity aspreviously described in A1.1.

In a second step a synthetic peptide corresponding to a CD8⁺ T cellepitope from ovalbumin was designed: in addition to the SIINFEKL (oneletter code for amino acid) sequence that is the precise epitopesequence, a cysteine residue with an activated Nitro-pyridin-sulfonylthiol group (Cys-NPys) was added at the N-terrninus of the peptideduring chemical synthesis. The activated Npys-cysteine was separatedfrom the SIINFEKL sequence by a flexible GGA motif to facilitate furtherproteolytic processing of the peptide within APC. The peptide(Cys-Npys-OVA, molecular weight: 1405 da) was synthesized by Neosystem(Strasbourg, France).

In a third step the Cys-Npys-OVA peptide was coupled to ACTM235 usingthe following procedure.

Ten mg of purified ACTM235 in 8 M urea, 20 mM Hepes-Na, pH 7.5, werereduced by incubation for 12 hrs in the presence of 10 mM Dithiothreitol(DTT. The efficiency of reduction was checked by an SDS-Page analysisunder non reducing conditions: essentially all the ACTM235 proteinmigrated as a single band of 200 kDa corresponding to the monomericspecies without evidence of any dimeric species (molecular weight ofabout 400 kDa). The reduced protein was then loaded on a DEAE-sepharose(Amersham Pharmacia Biotech) column (5 ml packed resin) equilibrated in8 M urea, 20 mM Hepes-Na, pH 7.5. The ACTM235 protein was fully retainedon the DEAE-sepharose resin that was then extensively washed with 8 Murea, 20 mM Hepes-Na, pH 7.5, 0.1 M NaCl (>100 ml) to remove any tracesof DTT (the absence of residual DTT was checked using the classicalEllman's reaction with 5,5′-Dithio-bis-(2-nitrobenzoic acid), DTNB). Thereduced ACTM235 protein was then eluted from the DEAE-sepharose resin in7 ml of 8 M urea, 20 mM Hepes-Na, pH 7.5, 0.5 M NaCl. Three mg (about 2μmol) of Cys-Npys-OVA peptide were then added to the reduced ACTM235protein (8 mg, about 45 nmol) and the mixture was incubated for 16 hrsat room temperature. Then, 25 ml of 20 mM Hepes-Na, pH 7.5 and 8 ml of 5M NaCl were added and this diluted mixture was then loaded on aPhenyl-Sepharose (Amersham Pharmacia Biotech) column (10 ml packedresin) equilibrated in 20 mM Hepes-Na, pH 7.5, 1 M NaCl. ThePhenyl-Sepharose resin was washed with 50 ml of 20 mM Hepes-Na, pH 7.5,1 M NaCl and then with 50 ml of 20 mM Hepes-Na, pH 7.5. The derivatizedACTM235 protein was then eluted in 8 M urea, 20 mM Hepes-Na, pH 7.5. Thetoxin concentration was determined spectrophotometrically from theabsorption at 280 nm using a molecular extinction coefficient of 142,000M-⁻¹.cm-⁻¹.

The Cys-Npys-OVA peptide was coupled using the same procedure to asecond recombinant CyaA toxin, CyaAE5-LCMVgp, which is a detoxifiedvariant (i.e. lacking the enzymatic activity as a result of the geneticinsertion of 2 amino acid LQ between residues 188 and 189). This toxinalso contains a 15 amino acid long polypeptide sequence(PASAKAVYNFATCGT) inserted between residues 224 and 225 of CyaA and thatcontains a single Cys residues. The plasmid encoding this recombinanttoxin is a derivative of pCACT-ova-E5 (Guermonprez et al. 2000) modifiedby the insertion between the Stul and Kpnl restriction sites of anappropriate synthetic double stranded oligonucleotide encoding thePASAKAVYNFATCGT sequence. The CyaAE5-LCMVgp protein was expressed andpurified as described previously in A.1.1.

The peptides shown in table 1 were also coupled similarly to anotherdetoxified recombinant CyaA toxin, CyaAE5-CysOVA, which contains thesame LQ dipeptide insertion in the catalytic site and a 14 amino acidsequence inserted between residues 224 and 225 of CyaA. This insertcontains a Cys residue adjacent to the OVA epitope as shown in FIG. 12.The plasmid encoding this recombinant toxin is a derivative ofpCACT-ova-E5 modified by the insertion between the BsiWl and Kpnlrestriction sites of an appropriate synthetic double strandedoligonucleotide encoding the ASCGSIINFEKLGT sequence. The CyaAE5-CysOVAprotein was expressed and purified as described previously in A.1.1 .

The CyaAE5-CysOVA can be considered as a general detoxified vector forchemical coupling of CTL epitopes by disulfide bridges. The presence ofthe OVA epitope within CyaAE5-CysOVA allows for an easy in vitro assayfor functionality in epitope delivery by measuring the presentation ofthe OVA epitope to specific T-cell hybridoma as described previously inB.1.4. TABLE 1 NPys CTL peptides coupled to CyaAE5-CysOVA Name ofepitopes Amino-acid sequence of peptides CEA 571 Cys(NPys)-GGYLSGANLNLGp100 Cys(NPys)-GGITDQVPFSV MelanA Cys(NPys)-GGEAAGIGILTV TyrosinaseCys(NPys)-GGYMDGTMSQV

Alternatively, the thiol groups of recombinant CyaA toxins can beactivated with 2,2′-dithiodipyridine (Sigma) and derivatised withpeptides containing a reduced cysteine (the procedure to reduce the Cysin synthetic peptides is provided by the manufacturer). This would beespecially appropriate if the desired peptide contains an internalcysteine residue.

C.2 Analysis of the in Vitro and in Vivo Immunogenicity of the OVAEpitope Chemically Coupled to CyaA

In vitro delivery of the OVA epitope to MHC class I molecules by CyaAafter chemical (CyaA-gp-S-S-OVA E5) or genetic coupling (CyaA-OVA E5)was first analysed by studying the presentation of these molecules tothe anti-OVA B3Z CD8⁺ T cell hybridoma by splenocytes. 3.10⁵ spleencells from C57BL/6 mice were co-cultured for 18 h with 105 B3Z cells inthe presence of various concentrations of the recombinant CyaA. The IL-2release by B3Z was measured in a CTLL proliferation assay.

As shown on the FIG. 13, a high IL-2 secretion was observed when B3Z wasstimulated either with CyaA-gp-S-S-OVA E5 or CyaA-OVA E5, thusdemonstrating that the OVA epitope chemically linked to CyaA was asefficiently delivered to the cytosolic pathway of the antigen-presentingcells than after genetic coupling. No IL-2 production was observed whenB3Z was stimulated with CyaA molecules lacking the OVA epitope, thusshowing the specificty of the IL-2 production.

In a second step, in vivo capacity of these molecules to induceOVA-specific CTL responses was then analysed. C57BL/6 mice (2 per group)were immunized by i.v. injection of 50 μg of the various CyaA Seven dayslater, 25.10⁶ spleen cells of individual mice were restimulated with 0.1μg of OVA peptide in the presence of 25.10⁶ irradiated syngenic spleencells for five days. The cytotoxic activity of the effector cells wasmeasured on 51 Cr-labeled EL4 target cells pulsed or not with 50 μM ofthe OVA peptide.

As shown in FIG. 14, high CTL responses were induced in mice immunizedby either CyaA-gp-S-S-OVA E5 or CyaA-OVA E5, thus demonstrating thecomparable efficacy of chemical or genetic coupling of the OVA epitope.No CTL response was observed in mice immunized with the control CyaAmolecules lacking the OVA epitope or when uncoated EL4 were used astarget cells, thus showing the specificity of the CTL responsesobserved.

In conclusion, these results clearly demonstrate that a CD8⁺ T cellepitope chemically linked to CyaA-derived proteinaceous vectors is veryefficiently delivered to the cytosolic pathway for MHC class Ipresentation and induces strong CTL response in vivo.

D. Identification of a CyaA-derived Fragment that Binds to CD11b/CD18and can be Used as Efficient Molecule Delivery Vectors

As an attempt to map the region that is involved in the interactionbetween CyaA and the CD11b/CD18 receptor, various subfragments of CyaAwere constructed and tested for their ability to compete with abiotinylated CyaA toxin for binding to CD11b/CD18 on the surface oftransfected CHO cells, using methods previously described in paragraphA.

D.1 Expression of CyaA373-1706

CyaA373-1706 protein was produced in E.coli by using a novel expressionvector pTRAC-373-1706 (FIG. 16). It is a derivative of plasmid pDL1312(Ladant 1995), constructed by replacing the neurocalcin gene of plasmidpDL1312 by the cyaC gene (that encodes cyaC which is involved in theconversion of proCyaA into the active toxin by post-translationalpalmitoylation of Lys 860 and 983 of CyaA) and the 3′ part of the cyaAgene that encodes the C-terminal domain—codons 373 to 1706- of CyaA (seeFIG. 16). Both cyaC and ‘cyaA genes are placed in the sametranscriptional unit under the control of the A phage Pr promoter. The3′ end of the cyaC gene was modified to introduce before its stop codon,a ribosome binding site to enhance the translation initiation of thedownstream ‘cyaA gene (FIG. 16). The resulting modification of the cyaCpolypeptide (the last 3 amino acid Gly-Thr-Ala at the C-terminus of cyaCwere replaced by the Asn-Arg-Glu-Glu sequence) had no effect on itsability to acylate CyaA. The 5′ end of the cyaA gene (coding for thecatalytic domain of the toxin (upstream of the unique BstBl site ofcyaA) was deleted and replaced by an appropriate synthetic doublestranded oligonucleotide encoding the MGCGN sequence (FIG. 16).

The pTRAC-373-1706 also encodes the thermosensitive A repressor C1⁸⁵⁷that strongly represses gene transcription at the λ Pr promoter attemperatures below 32° C., the origin of replication of colE1 and thebeta-lactamase gene that confers ampicillin resistance.

Expression of the CyaA373-1706 protein was carried out in E.coli strainBLR. Cells transformed with pTRAC-373-1706 were grown at 30° C. in LBmedium containing 150 mg/L of ampicillin until mid-log phase and thensynthesis of cyaC and of the truncated CyaA was induced by increasingthe growth temperature to 42° C. Bacteria were harvested after 3-4 hrsof further growth at 42° C. CyaA373-1706 protein was then purified asdescribed for the wild type CyaA (Guermonprez et al. 2000). CyaA373-1706is devoid of cAMP synthesizing activity, it exhibits hemolytic activityof sheep erythrocytes and contains also a unique cysteine residues inits MGCGN N-terminal sequence.

D2 Inhibition of CyaA-E5 Binding to CD11b by CyaA-E5 and CyaA fragments:1-383 (Catalytic Domain) and 373-1706 (Hydrophobic and Repeat Domains)

As shown on FIG. 15, a strong inhibition of biotinylated CyaA-E5 bindingwas achieved after incubation of transfected CHO cells with CyaA-E5. Asimilar inhibition was obtained after incubation of these cells with thefragment CyaA-373-1706 whereas CyaA 1-383 had no significant effect onthe binding of the biotinylated CyaA-E5.

Thus, these results clearly demonstrate that the fragment of CyaA thatencompasses residues 373 to 1706, CyaA373-1706, contains the structuresessentially required for the interaction of CyaA with the CD11b/CD18receptor.

E. Conclusions

Since MHC class I molecules usually present peptides derived fromendogenously synthesized proteins, epitopes must be delivered to thecytosol of antigen presenting cells to elicit CTL responses. It has beenpreviously established that the CTL priming activity of recombinant ACTprotein relies at least in part on its ability to deliver CD8⁺ T cellepitopes into the cytosol of antigen-presenting cells (APC). In oneapproach, epitopes were genetically inserted within the catalytic domainof CyaA as it is known that, for the wild-type toxin, this part of thepolypeptide reaches the cytosol of target cells where it exerts itstoxic effect (Le. cAMP synthesis). After entering the cells, the CyaAcatalytic domain harboring the epitope insert must be proteolyticallyprocessed to release the matured CD8⁺ T cell epitope which then will betranslocated to the endoplasmic reticulum to associate with MHC class Imolecules.

This proteolytic processing, carried out by the proteasome, might be alimiting step in numerous cases (Pamer and Cresswell, 1998; Cascio etal., 2001), leading to a significant decrease of the overall yield ofthe mature epitope. The alternative approach, described here, thatconsist in the linkage of the epitopes to the catalytic domain of CyaAby means of a disulfide bond, offers a significant advantage in that,with this design, matured epitopes (requiring only N-terrninal triming)will be quantitatively released from CyaA inside the cytosol of APC. Inaddition, this should be a very versatile system as a single CyaAcarrier protein could be easily and rapidly coupled to any desiredsynthetic peptide. Furthermore, it will be easy to introduce by geneticengineering, several cysteine residues within the catalytic domain (suchas in previously mapped permissive sites, WO 93/21324 (INSTITUTPASTEUR)) of a recombinant detoxified CyaA toxin so that multiplepeptides can be coupled on the same CyaA molecule. This should increasethe overall epitope delivery and immunogenicity of the recombinantprotein.

Deletion mapping allowed to identify the C-terminal part of CyaA (aa373-1706) as the region that is involved in the interaction of the toxinwith its CD11b/CD18 receptor. The truncated protein CyaA373-1706 can betherefore used as a protein module to target CD11b+ cells in vivo. Inparticular, polypeptides or proteins corresponding to antigens ofinterest can be genetically fused to CyaA373-1706 to be delivered todendritic cells in order to elicit specific immune responses. Similarcoupling can be performed on the recombinant CyaA373-1706 protein thatalso contained a unique cysteine at its N terminal end.

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1-56. (canceled)
 57. The method of modifying CD11b expressing cells,wherein the method comprises targeting a molecule of interest to thecells by contacting a Bordetella adenylcyclase with cells, wherein theBordetella adenylcyclase comprises the molecule of interest.
 58. Themethod of claim 57, wherein the targeting of the molecule of interest iseffective in vivo.
 59. The method of claim 57, wherein the molecule ofinterest is translocated to the cytosol of the CD11b expressing cells.60. The method of claim 57, wherein the CD11b expressing cells comprisesdendritic cells.
 61. The method of claim 57, wherein the CD11bexpressing cells comprises myeloid dendritic cells.
 62. The method ofclaim 57, wherein the CD11b expressing cells comprise neutrophils. 63.The method of claim 57, wherein the adenylcyclase comprises agenetically modified Bordetella species adenylcyclase.
 64. The method ofclaim 57, wherein the adenylcyclase comprises a non-toxic form.
 65. Themethod of claim 57, wherein the adenylcyclase comprises a Bordetellapertussis adenylcyclase.
 66. The method of claim 57, wherein themolecule of interest is selected from peptides, glycopeptides,lipopeptides, polysaccharides, oligosaccharides, nucleic acids, lipids,and chemicals.
 67. The method of claim 66, wherein the molecule ofinterest comprises an antigen.
 68. The method of claim 66, wherein themolecule of interest comprises an epitope.
 69. The method of claim 66,wherein the molecule of interest comprises a heterologous peptide fusedto the N-terminal extremity of a genetically modified Bordetellaadenylcyclase lacking all or part of its N-terminal catalytic domain.70. The method of claim 69, wherein the molecule of interest comprises aheterologous peptide fused to the N-terminal extremity of a geneticallymodified Bordetella pertussis adenylcyclase lacking residues 1-373. 71.The method of claim 67, wherein said antigen is selected from anintracellular bacterial cell antigen, a tumoral cell antigen, a viralantigen, a fungus antigen, and a parasite cell antigen.
 72. The methodof claim 71, wherein said antigen is selected from a poliovirus antigen,an HIV antigen, an influenza virus antigen, a choriomeningitis virusepitope, and a tumor antigen.
 73. The method of claim 57, wherein themolecule of interest comprises a drug chemically or genetically coupledto the adenylcyclase toxin.
 74. The method of claim 73, wherein the drugis chemically coupled by a disulfide bond to a genetically insertedcysteine residue located in a catalytic domain of the adenylcyclase. 75.The method of claim 74, wherein the adenylcyclase is a Bordetellapertussis adenylcyclase, and wherein the cysteine residue is inserted ata site selected from residues 137-138, 224-225, 228-229, 235-236,317-318, and 335-336.
 76. The method of any one of claims 73-75, whereinthe drug is an anti-inflammatory drug.
 77. The method of claim 57,wherein the adenylcyclase comprises the molecule of interest in itscatalytic domain.
 78. The method of claim 77, wherein the molecule ofinterest is inserted at a permissive site within the catalytic domain.79. The method of claim 78, wherein the adenylcyclase comprises aBordetella pertussis adenylclase, and wherein the catalytic domaincomprises amino acids 1-400.
 80. A method of modifying CD11b expressingcells, wherein the method comprises targeting a molecule of interest tothe cells by administering to a mammal a Bordetella adenylcyclase,wherein the Bordetella adenylcyclase comprises the molecule of interest.81. The method of claim 80, wherein the CD11b expressing cells comprisemyeloid dendritic cells.
 82. The method of claim 80, wherein theadenylcyclase is a Bordetella pertussis adenylcyclase
 83. The method ofclaim 80, wherein the molecule of interest comprises a drug chemicallycoupled to the adenylcyclase toxin by a disulfinde bond to a geneticallyinserted cysteine residue inserted at a site selected from residues137-138, 224-225, 228-229, 235-236, 317-318, and 335-336.
 84. The methodof claim 80, wherein the CD11b expressing cells comprise myeloiddendritic cells, wherein the adenylcyclase is a Bordetella pertussisadenylcyclase, wherein the molecule of interest comprises a drugchemically coupled to the adenylcyclase toxin by a disulfinde bond to agenetically inserted cysteine residue inserted at a site selected fromresidues 137-138, 224-225, 228-229, 235-236, 317-318, and 335-336.